Mycobacterium Tuberculosis: Molecular: Infection Biology, Pathogenesis, Diagnostics and New Interventions

Seyed
Ehtesham Hasnain
Nasreen Z. Ehtesham · Sonam Grover
Editors
Mycobacterium
tuberculosis: Molecular
Infection Biology,
Pathogenesis, Diagnostics
and New Interventions
Mycobacterium tuberculosis: Molecular
Infection Biology, Pathogenesis, Diagnostics
Seyed Ehtesham Hasnain •
Nasreen Z. Ehtesham • Sonam Grover
Editors
Mycobacterium
tuberculosis: Molecular
Infection Biology,
Pathogenesis, Diagnostics
Editors
Seyed Ehtesham Hasnain Nasreen Z. Ehtesham
JH Institute of Molecular Medicine, Inflammation Biology and Cell Signaling
Jamia Hamdard Laboratory
New Delhi, Delhi, India ICMR-National Institute of Pathology,
Safdarjung Hospital Campus
Dr Reddy’s Institute of Life Sciences
New Delhi, Delhi, India
University of Hyderabad Campus
Hyderabad, India
Sonam Grover
JH Institute of Molecular Medicine,
Jamia Hamdard
ISBN 978-981-32-9412-7 ISBN 978-981-32-9413-4 (eBook)

https://doi.org/10.1007/978-981-32-9413-4
# Springer Nature Singapore Pte Ltd. 2019

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Preface
Tuberculosis (TB), the disease, and Mycobacterium tuberculosis (M.tb), the causa-
tive pathogen, need no introduction. This book on TB will serve as a comprehensive
knowledge base for M.tb, as well as nontubercular bacteria (NTM).
Presenting a wealth of information on the molecular infection biology of M.tb, as
well as nontuberculous mycobacteria (NTM), this book provides an overview of the
functional role of the PE/PPE group of proteins, which is exclusive to the genus
Mycobacterium, of host-pathogen interactions, and of virulence. It also explores the
pathogenesis of the infection, pathology, epidemiology, and diagnosis of NTM. Last
but not least, it discusses current and novel approaches in vaccine development against
tuberculosis, including the role of nanotechnology. This book highlights developments
in understanding the disease at the molecular levels and identification and validation of
molecular drug targets, molecular epidemiology, and novel diagnostic techniques.
With state-of-the-art contributions from experts in the respective domains, this volume
will be an informative resource for current practitioners as well as medical students,
postgraduates, and researchers and is expected to be much in demand. The book is
mainly divided into three sections covering all major aspects of TB.
Pathogenesis and Epidemiology of Tuberculosis
This section starts with a chapter on the history of tuberculosis with a description of
fascinating events starting from the discovery of the elusive TB bacteria. A chapter
on TB comorbidities describes the explicit involvement of TB in promoting other
lung diseases such as bronchiectasis and chronic obstructive pulmonary disease
during childhood. To reduce the global burden of chronic lung diseases, the
management of TB in the early stages is imperative. A chapter on the role of host
factors in susceptibility to TB serves as a primer to understand the efforts mounted
by the host which can be useful in designing host-directed therapies and also reduce
tissue damage due to chronic TB infections. A chapter on extrapulmonary tuber-
culosis (EPTB) provides an in-depth overview of the clinical diagnosis and treatment
of the different forms of EPTB. This section also covers nontubercular mycobacteria
(NTM), a high priority due to its misdiagnosis and association with cystic fibrosis,
COPD, and nosocomial infections. The last chapter in this section discusses the
v
vi Preface
spread of M.tb strains circulating in New Zealand and emphasizes the need for early
detection and treatment of active cases together with contact tracing, if one has to
minimize the emergence of epidemiological clusters of the disease. This chapter also
illustrates the importance of a political will and decisive legislation in bringing down
the incidence of disease from 150 cases per 100,000 to less than 10 cases per
100,000 and steps a country need to take to avoid new cases of TB.
Molecular and Infection Biology of M.tb
M.tb is highly adapted for survival in the extremely hostile intracellular environment
in host macrophages. In order to cope up with stresses encountered by the bacterium
in macrophages, it modulates its gene expression in various ways. This section is a
combination of chapters where molecular aspects of M.tb have been highlighted to
understand bacterial survival strategy. This section begins with a chapter on
methyltransferases, present in large number in M.tb, modifying DNA, RNA,
proteins, lipids, and other biomolecules of both pathogen and host and illustrates
how these epigenetic modifiers impact virulence and pathogenesis of M.tb. Three
consecutive chapters provide a comprehensive overview on functional biology of the
unique group of proteins called PE/PPE, which is present exclusively in the genus
Mycobacterium and nowhere else in the living kingdom. The discussions include
evolutionary developments, characterization, and transcriptional regulation of the
PE/PPE family along with their involvement in host immunomodulation and also
showcase the functional role of intrinsically disordered regions concentrated within
this protein family. A chapter on the importance of cell wall-associated poly-α-L-
glutamine in the biology of pathogenic mycobacteria is also presented in this book
which dissects the unique cell wall architecture of M.tb contributing to the unre-
stricted success of the bacterium during infection. Cellular stress responses during
M.tb infection have been highlighted in the next chapter including the strategies
adopted by M.tb to influence the host innate and adaptive immune responses. The
role of heat shock proteins in the mycobacterial pathogenesis has also been discussed
in a separate chapter. The importance of endoplasmic reticulum (ER) stress in M.tb.
pathogenesis including the ER stress pathways evoked by M.tb proteins such as
ESAT-6, HBHA, 38-kDa antigen, and PE_PGRS5 is nicely presented in the next
chapter. This is followed by a discussion on the role of toxin-antitoxin (TA) systems
in bacterial pathogenesis and the abundance of such TA systems in M.tb, which in
turn helps the pathogen fight against various cellular stresses including acidic,
hypoxic, and oxidative and also challenges posed by the immune system. A detailed
description of the nucleotide excision repair (NER) pathway in mycobacteria is in a
separate chapter where in-depth studies of the structure-function relationship of
DNA repair throw light on the molecular mechanisms employed by M.tb for self-
defense. Such studies will enable the development of novel therapeutic interventions
against TB.
Preface vii
The chapter on mesenchymal stem cells (MSC) describes its potential in M.tb
persistence, resuscitation, and disease reactivation and highlights studies on MSCs
as a reservoir of M.tb, which could be a probable target for developing new
therapeutics against non-replicating bacteria and viable but non-culturable bacteria.
The last chapter in this section emphasizes biofilm and its importance in drug
resistance and how biofilm disruption could be an important strategy not only to
contain the TB bacteria but also to reduce IC50 of antitubercular drugs.
Vaccines, Diagnostics, and New Interventions
This section begins with a detailed description of the best practices for mycobacteria
research laboratories, given the deadly nature of this airborne pathogen. It also
covers the occupational risks of working with M.tb and identifies the primary and
secondary barriers to M.tb exposure, controls that reduce the risk of exposure and
also the good practices and guidelines to be followed during experimental manipu-
lation so as to avoid getting infected with the bacterium. Of particular interest are the
deployment of biosafety cabinets, good waste management practices, spills manage-
ment, and of course occupational health programs and facility decontamination. This
is followed by comprehensive review on TB vaccines. The presently used whole cell
vaccine such as BCG is inefficient in TB-endemic population. Thus, there is an
urgent need for novel vaccines capable of providing complete protection in all age
groups as well as in different stages of infection. This is followed by a chapter
describing the immunotherapeutic properties of Mycobacterium indicus pranii
(MIP) and its likely use as an alternative vaccine candidate. MIP, a nonpathogenic
saprophytic mycobacterium, shares many cross-reactive antigens with M.tb and
proved to be effective in category II TB as an adjunct to MDT.
History as well as present status of TB diagnostics has also been covered in a
chapter entitled “TB Diagnostics: Journey from Smear Microscopy to Whole
Genome Sequencing.” Developing point of care diagnostic with fast, reliable, and
affordable features is vital to curb and control TB and HIV-TB burden in resource-
poor countries. This is discussed in the chapter that also focusses on the journey of
such diagnostic techniques starting from older strategies such as smear microscopy
to modern-day techniques like whole genome sequencing.
An interesting chapter describes how to break the transmission of TB by bridging
the gap in controlling TB in endemic settings. In order to control the spread of
MDR-TB, there is a need to use better diagnostic tool and health measures to
interrupt pathogen transmission. Challenges and advances in TB drug discovery
are also discussed in detail including the advancement of methodologies for enhanc-
ing bioavailability of drugs by different means such as nanoparticle-/liposome-
mediated delivery systems and devices for sustained release.
The last chapter in this section is based on the experience of actual drug discovery
gained by the researchers at Johns Hopkins University. While describing the pro-
cesses involved in TB drug discovery, and a review of existing candidate drugs, the
authors illustrate their efforts in using drug repurposing strategy for development of
viii Preface
new drugs against MDR and XDR strains of M.tb. The very powerful carbapenem
antibiotic, belonging to the beta-lactam group, has been used as a model new drug. In
addition, the use of antisense RNA-based therapeutics and CRISPER-CAS system-
based new interventions are also discussed.
New Delhi, Delhi, India Seyed Ehtesham Hasnain

Nasreen Z. Ehtesham
Sonam Grover
Acknowledgments
This book is made possible, thanks to the unqualified support, hard work and
adherence to the deadlines by scientists, clinicians, and academicians from around
the globe who took their time off from their other responsibilities to present new
knowledge about TB, a disease that largely affects the underprivileged in underde-
veloped countries. We are indeed honored to be associated with renowned
personalities, working on different aspects of TB, and are humbled by their commit-
ment, kindness, and endurance.
We do hope that this book will add new knowledge about the unique attributes of
this super smart bacteria so as to enable the development of new interventions
including drugs and vaccines and also point-of-care, rapid, and cost-effective diag-
nosis of TB.
The contributions of our reviewers who generously donated their time to review
every chapter are gratefully acknowledged. We would like to put on record the
efforts of Dr. Soumya Swaminathan, former Director General, Indian Council of
Medical Research and Secretary, Department of Health Research, presently Chief
Scientist, WHO Geneva, for writing the foreword which elegantly and succinctly
summarizes the salient features of the mammoth effort that have gone into compiling
this book.
We would like to duly acknowledge the editorial support provided by Ms. Ruby
Palta (PS to SEH) and Dr. Salma Jamal and Dr. Rishabh Gangwar, Postdocs in our
lab for their technical help. Springer Nature deserves full credit for their persistence,
professionalism, and enthusiasm during the publication of the book.
Finally, our respective families deserve all the credit for allowing us to bypass
family and social commitments during the completion of this project.
ix
Contents
Part I Pathogenesis and Epidemiology of Tuberculosis

1 History of TB: Robert Koch and Beyond . . . . . . . . . . . . . . . . . . . . 3
Ashfaq Hasan
2 Tuberculosis as an Underlying Etiological Factor
for Other Human Respiratory Diseases . . . . . . . . . . . . . . . . . . . . . . 17
Ronan F. O’Toole
3 Host Factors in Tuberculosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Ruxana T. Sadikot
4 Extrapulmonary Tuberculosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Surendra K. Sharma and Alladi Mohan
5 Infections with Nontuberculous Mycobacteria: Increased
Awareness and Recent Developments . . . . . . . . . . . . . . . . . . . . . . . 55
Astrid Lewin and Hubert Schäfer
6 Tuberculosis in New Zealand: Historical Overview
to Modern Epidemiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
Ronan F. O’Toole
Part II Molecular and Infection Biology of M. tuberculosis

7 Mycobacterial Methyltransferases: Significance in Pathogenesis
and Virulence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
Sonam Grover, Rishabh Gangwar, Salma Jamal, Sabeeha Ali,
Khairun Nisaa, Nasreen Z. Ehtesham, and Seyed Ehtesham Hasnain
8 The PE and PPE Family Proteins of Mycobacterium
tuberculosis: What they Are Up To? . . . . . . . . . . . . . . . . . . . . . . . . 123
Ravi Pal, Faiza Nazar, and Sangita Mukhopadhyay
9 Intrinsically Disordered Regions in PE/PPE Protein Family
of Mycobacterium tuberculosis: Moonlighting Function . . . . . . . . . . 151
Farha Naz, Javeed Ahmad, Mohd Shariq, Mohd Arish,
Javaid A. Sheikh, Seyed E. Hasnain, and Nasreen Z. Ehtesham
xi
xii Contents
10 Comparative In Silico Analyses Reveal Crucial Factors

for Virulence, Antigenicity, and Evolution in M.tb . . . . . . . . . . . . . . 171
Yadvir Singh
11 Importance of Cell Wall-Associated Poly-α-L-Glutamine
in the Biology of Pathogenic Mycobacteria . . . . . . . . . . . . . . . . . . . 189
Rajni Garg, Rajesh Mani, Manish Gupta, Deeksha Tripathi,
Harish Chandra, Rakesh Bhatnagar, and Nirupama Banerjee
12 Cellular Stress Responses and Immunological Regulations
During Mycobacterium tuberculosis Infection . . . . . . . . . . . . . . . . . . 203
Nooruddin Khan, Gillipsie Minhas, K. Kala jyothi, and Jyoti Sharma
13 Heat Shock Proteins in the Pathogenesis of Mycobacterium
tuberculosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221
Prajna Tripathi and Janendra K. Batra
14 Endoplasmic Reticulum Stress: Importance in Pathogenesis
of Mycobacterium tuberculosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241
Tarina Sharma, Sonam Grover, Nasreen Z. Ehtesham,
and Seyed E. Hasnain
15 Toxin-Antitoxin (TA) Systems in Stress Survival
and Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257
Ashutosh Kumar, Anwar Alam, Pranami Bharadwaj,
Sharmistha Tapadar, Mamta Rani, and Seyed E. Hasnain
16 Nucleotide Excision Repair Pathway in Mycobacteria . . . . . . . . . . . 275
Manoj Thakur and K. Muniyappa
17 Mesenchymal Stem Cells: A Hidden Arsenal for
Mtb Persistence, Resuscitation, and Reactivation . . . . . . . . . . . . . . 301
Jaishree Garhyan, Bikul Das, and Rakesh Bhatnagar
18 Biofilms: A Phenotypic Mechanism of Bacteria Conferring
Tolerance Against Stress and Antibiotics . . . . . . . . . . . . . . . . . . . . . 315
Anwar Alam, Ashutosh Kumar, Prajna Tripathi,
Nasreen Z. Ehtesham, and Seyed E. Hasnain
Part III Vaccines, Diagnostics and New Interventions

19 Best Practices in Mycobacterial Research Laboratories . . . . . . . . . 337
Noman Siddiqi
20 Clinical Aspects and Principles of Management
of Tuberculosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355
Ashfaq Hasan, Sai Haranath Praveen, Chandrakant Tarke,
and Fahad Abdullah
Contents xiii
21 Tuberculosis Vaccine: Past Experiences and Future Prospects . . . . 375

Gurpreet Kaur, Deepjyoti K. Das, Sanpreet Singh, Junaid Khan,
Mohammad Sajid, Hilal Bashir, Mohammad Aqdas, Shikha Negi,
Uthaman Gowthaman, and Javed N. Agrewala
22 Immunotherapeutic Potential of Mycobacterium indicus pranii
Against Tuberculosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 407
Sangeeta Bhaskar and Bindu Singh
23 TB Diagnostics: Journey from Smear Microscopy
to Whole Genome Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 419
Himanshu Vashistha and K. K. Chopra
24 Breaking the Transmission of TB: A Roadmap to Bridge
the Gaps in Controlling TB in Endemic Settings . . . . . . . . . . . . . . . 451
Pooja Singh, Jasmine Samal, Sheeba Zarin, Ravikrishnan Elangovan,
Seyed E. Hasnain, and Nasreen Z. Ehtesham
25 Challenges and Advances in TB Drug Discovery . . . . . . . . . . . . . . . 463
Garima Khare, Prachi Nangpal, and Anil K. Tyagi
26 Repurposing of Carbapenems for the Treatment
of Drug-Resistant Tuberculosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 497
Pankaj Kumar, Urvashi B. Singh, Gyanu Lamichhane,
and Elizabeth Story-Roller
About the Editors
Prof. Seyed Ehtesham Hasnain has contributed significantly to tuberculo-

sis (TB) research, focusing on infection biology and functional epidemiology. He is
Fellow of Robert Koch Institute (Berlin), American Academy of Microbiology (USA),
TWAS, and all major Indian Science Academies (FNA/FASc/FNASc) and member of
German National Academy of Sciences Leopoldina. He has received numerous
recognitions and honors, including Alexander von Humboldt Research Award
(Germany), Shanti Swarup Bhatnagar Prize, GD Birla Award, JC Bose Fellowship
Award, and many others. A former member of the Science Advisory Council to the
Prime Minister of India for two terms (2004–2014), he was the first director of the
Centre for DNA Fingerprinting and Diagnostics, Hyderabad, and later vice-chancellor
of the prestigious University of Hyderabad (2005–2011) and served as invited profes-
sor at the IIT Delhi (2011–2019). Currently, he is the Vice-Chancellor of Jamia
Hamdard and also professor at JH Institute of Molecular Medicine, where he leads a
very large, active, and generously funded TB research group.
Dr. Nasreen Z. Ehtesham received her Master’s degree from Aligarh Muslim
University, Aligarh, and graduate training from the University of Alberta,
Edmonton, Canada. She pursued her Ph.D. at the National Institute of Immunology,
New Delhi (1991), and joined the ICGEB as a Rockefeller postdoc fellow. She was
appointed as Deputy Director, National Institute of Nutrition, Hyderabad, where her
group focused on nutrition and inflammation biology. She established the link
between pathogenesis, inflammation, and stress response using the Mycobacterium
tuberculosis infection model. An elected Fellow of the National Academy of
Sciences, she has served as a member of several decision-making bodies of the
Health and S&T ministries. She is the recipient of many awards, including the ICMR
Kshanika Oration Award for her contributions to tuberculosis biology. She has
published over 70 research papers in prestigious journals such as PNAS and serves
on the editorial boards of a number of journals. Currently, she is Director-in-Charge
of the ICMR National Institute of Pathology, New Delhi.
Dr. Sonam Grover is a UGC Assistant Professor at JH Institute of Molecular

Medicine, Jamia Hamdard, New Delhi. She completed her Ph.D. at JNU (2014)
and joined Kusuma School of Biological Sciences, IIT Delhi, as a postdoctoral
xv
xvi About the Editors
fellow in the same year. During this tenure, she received a Women Scientist Grant
from the Department of Health Research, Government of India. Her main areas of
research include Mycobacterium tuberculosis host-pathogen interactions, drug
repurposing, and identifying novel drug targets against tuberculosis. She has
published over 25 research papers in various international peer-reviewed journals
including mBio and Molecular Neurobiology. She has delivered talks and presented
her work at several national and international conferences and symposia. She is PI/
co-PI in research projects funded by agencies such as DBT and DHR. She was also
awarded the prestigious “ASCB Travel Award for Graduate Students” for presenting
her research at international conferences.
Contributors
Fahad Abdullah Department of Respiratory Medicine, Deccan College of Medical

Sciences, Hyderabad, India
Javed N. Agrewala Immunology Laboratory, Institute of Microbial Technology,
Chandigarh, India
Centre for Biomedical Engineering, Indian Institute of Technology-Ropar,
Rupnagar, India
Javeed Ahmad Inflammation Biology and Cell Signaling Laboratory,
ICMR-National Institute of Pathology, Safdarjung Hospital Campus, New Delhi,
Delhi, India
Anwar Alam JH Institute of Molecular Medicine, Jamia Hamdard, New Delhi,
Delhi, India
Inflammation Biology and Cell Signaling Laboratory, ICMR-National Institute of
Pathology, Safdarjung Hospital Campus, New Delhi, Delhi, India
Mohammad Aqdas Immunology Laboratory, Institute of Microbial Technology,
Chandigarh, India
Mohd Arish JH Institute of Molecular Medicine, Jamia Hamdard, New Delhi,
Delhi, India
Nirupama Banerjee Molecular Biology and Genetic Engineering Laboratory,
School of Biotechnology, Jawaharlal Nehru University, New Delhi, Delhi, India
Hilal Bashir Immunology Laboratory, Institute of Microbial Technology,
Chandigarh, India
Janendra K. Batra National Institute of Immunology, New Delhi, Delhi, India
Department of Biochemistry, School of Chemical and Life Sciences, Jamia
Hamdard, New Delhi, Delhi, India
Pranami Bharadwaj Department of Microbiology, Tripura University (A Central
University), Agartala, Tripura, India
Sangeeta Bhaskar Product Development Cell-I, National Institute of Immunology,
xvii
xviii Contributors
Rakesh Bhatnagar Molecular Biology and Genetic Engineering Laboratory,

School of Biotechnology, Jawaharlal Nehru University, New Delhi, Delhi, India
Banaras Hindu University, Varanasi, Uttar Pradesh, India
Harish Chandra Department of Microbiology, School of Biomedical & Pharma-
ceutical Sciences, Babasaheb Bhimrao Ambedkar University, Lucknow, India
K. K. Chopra New Delhi Tuberculosis Center, New Delhi, Delhi, India
Bikul Das Department of Stem Cell and Infectious Diseases, Kavikrishna Labora-
tory, Guwahati Biotech Park, Indian Institute of Technology, Guwahati, India
Department of Stem Cells and Infectious Diseases, Thoreau Laboratory for Global
Health, M2D2, University of Massachusetts, Lowell, MA, USA
Deepjyoti K. Das Immunology Laboratory, Institute of Microbial Technology,
Chandigarh, India
Nasreen Z. Ehtesham Inflammation Biology and Cell Signaling Laboratory,
Delhi, India
Ravikrishnan Elangovan Department of Biochemical Engineering and Biotech-
nology, Indian Institute of Technology-Delhi, New Delhi, Delhi, India
Rishabh Gangwar JH Institute of Molecular Medicine, Jamia Hamdard, New
Delhi, Delhi, India
Rajni Garg Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore,
Karnataka, India
Department of Microbiology and Cell Biology, Indian Institute of Science,
Bangalore, Karnataka, India
Jaishree Garhyan Department of Microbiology, University of Massachusetts,
Amherst, MA, USA
Molecular Biology and Genetic Engineering Laboratory, School of Biotechnology,
Jawaharlal Nehru University, New Delhi, Delhi, India
Uthaman Gowthaman Department of Laboratory Medicine, Immunobiology and
Orthopedics, Yale University School of Medicine, New Haven, CT, USA
Sonam Grover JH Institute of Molecular Medicine, Jamia Hamdard, New Delhi,
Delhi, India
Manish Gupta Molecular Biology and Genetic Engineering Laboratory, School of
Biotechnology, Jawaharlal Nehru University, New Delhi, Delhi, India
Ashfaq Hasan Department of Respiratory Medicine, Deccan College of Medical
Sciences, Hyderabad, India
Contributors xix
Seyed Ehtesham Hasnain JH Institute of Molecular Medicine, Jamia Hamdard,

Dr Reddy’s Institute of Life Sciences, University of Hyderabad Campus, Hyderabad,
India
Salma Jamal JH Institute of Molecular Medicine, Jamia Hamdard, New Delhi,
Delhi, India
K. Kala jyothi Department of Biotechnology and Bioinformatics, School of Life
Sciences, University of Hyderabad, Hyderabad, Telangana, India
Gurpreet Kaur Immunology Laboratory, Institute of Microbial Technology,
Chandigarh, India
Centre for Biomedical Engineering, Indian Institute of Technology-Ropar,
Rupnagar, India
Junaid Khan Immunology Laboratory, Institute of Microbial Technology,
Chandigarh, India
Nooruddin Khan Department of Biotechnology and Bioinformatics, School of
Life Sciences, University of Hyderabad, Hyderabad, Telangana, India
Garima Khare Department of Biochemistry, University of Delhi South Campus,
Ashutosh Kumar Department of Microbiology, Tripura University (A Central
Pankaj Kumar Department of Biochemistry, School of Chemical and Life
Sciences, Jamia Hamdard, Delhi, India
Gyanu Lamichhane Division of Infectious Diseases School of Medicine, Johns
Hopkins University, Baltimore, MD, USA
Astrid Lewin Robert Koch Institute, Unit 16 Mycotic and Parasitic Agents and
Mycobacteria, Berlin, Germany
Rajesh Mani Molecular Biology and Genetic Engineering Laboratory, School of
Biotechnology, Jawaharlal Nehru University, New Delhi, Delhi, India
Gillipsie Minhas Department of Biotechnology and Bioinformatics, School of Life
Alladi Mohan Department of Medicine, Sri Venkateswara Institute of Medical
Sciences, Tirupati, India
Sangita Mukhopadhyay Laboratory of Molecular Cell Biology, Centre for DNA
Fingerprinting and Diagnostics (CDFD), Hyderabad, Telangana, India
K. Muniyappa Department of Biochemistry, Indian Institute of Science,
Bangalore, India
xx Contributors
Prachi Nangpal Department of Biochemistry, University of Delhi South Campus,

Faiza Nazar Laboratory of Molecular Cell Biology, Centre for DNA Fingerprint-
ing and Diagnostics (CDFD), Hyderabad, Telangana, India
Farha Naz Inflammation Biology and Cell Signaling Laboratory, ICMR-National
Institute of Pathology, Safdarjung Hospital Campus, New Delhi, Delhi, India
Shikha Negi Immunology Laboratory, Institute of Microbial Technology,
Chandigarh, India
Khairun Nisaa Department of Life Sciences, Ben Gurion University of the Negev,
Beersheba, Israel
Ronan F. O’Toole School of Molecular Sciences, College of Science, Health and
Engineering, La Trobe University, Melbourne, VIC, Australia
Department of Clinical Microbiology, School of Medicine, Trinity College Dublin,
Dublin, Ireland
School of Medicine, College of Health and Medicine, University of Tasmania,
Hobart, Tasmania, Australia
Ravi Pal Laboratory of Molecular Cell Biology, Centre for DNA Fingerprinting
and Diagnostics (CDFD), Hyderabad, Telangana, India
Graduate Studies, Manipal Academy of Higher Education, Manipal, Karnataka,
India
Sai Haranath Praveen Department of Pulmonary and Critical Care Medicine,
Apollo Hospitals, Hyderabad, India
Mamta Rani Plant Microbe Interactions Laboratory, National Institute of Plant
Genome Research, New Delhi, Delhi, India
Sabeeha Ali Kusuma School of Biological Sciences, Indian Institute of Technol-
ogy, New Delhi, Delhi, India
Ruxana T. Sadikot Section of Pulmonary, Critical Care Medicine, Emory Univer-
sity, Atlanta, GA, USA
Mohammad Sajid Immunology Laboratory, Institute of Microbial Technology,
Chandigarh, India
Jasmine Samal Inflammation Biology and Cell Signaling Laboratory, ICMR-
National Institute of Pathology, Safdarjung Hospital Campus, New Delhi, Delhi,
India
Hubert Schäfer Robert Koch Institute, Unit 16 Mycotic and Parasitic Agents and
Mycobacteria, Berlin, Germany
Mohd Shariq Inflammation Biology and Cell Signaling Laboratory, ICMR-
National Institute of Pathology, Safdarjung Hospital Campus, New Delhi, Delhi,
India
Contributors xxi
Jyoti Sharma Department of Biotechnology and Bioinformatics, School of Life

Surendra K. Sharma JH Institute of Molecular Medicine, Jamia Hamdard,
Departments of General Medicine & Pulmonary Medicine, JNMC, Datta Meghe
Institute of Medical Sciences (DMIMS), Wardha, Maharashtra, India
Department of Internal Medicine (WHO Collaborating Centre for Research &
Training in Tuberculosis, Centre of Excellence for EPTB, MoH & FW, GoI), All
India Institute of Medical Sciences, New Delhi, Delhi, India
Tarina Sharma Molecular Infection and Functional Biology Lab, Kusuma School
of Biological Sciences, Indian Institute of Technology-Delhi, New Delhi, Delhi,
India
Javaid A. Sheikh Department of Biotechnology, School of Chemical and Life
Sciences, Jamia Hamdard, New Delhi, Delhi, India
Noman Siddiqi Harvard TH Chan School of Public Health, Boston, MA, USA
Bindu Singh Product Development Cell-I, National Institute of Immunology,
Pooja Singh JH Institute of Molecular Medicine, Jamia Hamdard, New Delhi,
Delhi, India
Sanpreet Singh Immunology Laboratory, Institute of Microbial Technology,
Chandigarh, India
Urvashi B. Singh Tuberculosis Section, Department of Microbiology, A.I.I.M.S.,
Yadvir Singh Department Molecular Biology, Max Planck Institute for Infection
Biology, Berlin, Germany
Elizabeth Story-Roller Division of Infectious Diseases, School of Medicine, Johns
Hopkins University, Baltimore, MD, USA
Sharmistha Tapadar Department of Microbiology, Tripura University (A Central
Chandrakant Tarke Department of Pulmonary and Sleep Medicine, Apollo
Hospitals, Hyderabad, India
Manoj Thakur Department of Biochemistry, Indian Institute of Science,
Bangalore, India
Deeksha Tripathi Department of Microbiology, School of Life Sciences, Central
University of Rajasthan, Ajmer, Rajasthan, India
Prajna Tripathi National Institute of Immunology, New Delhi, Delhi, India
JH Institute of Molecular Medicine, Jamia Hamdard, New Delhi, Delhi, India
xxii Contributors
Anil K. Tyagi Department of Biochemistry, University of Delhi South Campus,

Guru Gobind Singh Indraprastha University, New Delhi, Delhi, India
Himanshu Vashistha Viral Hepatitis Division, National Centre for Disease Con-
trol, New Delhi, Delhi, India
Sheeba Zarin Inflammation Biology and Cell Signaling Laboratory,
Delhi, India
Part I
Pathogenesis and Epidemiology of
Tuberculosis
History of TB: Robert Koch and Beyond
1
Ashfaq Hasan
That which is not expressed will be forgotten.

And what is forgotten will happen again.
Yevgeny Yevtushenko
The only thing we learn from history is that we learn nothing
from history.
Georg Wilhelm Friedrich Hegel
Abstract
Tuberculosis is a disease like no other. It has claimed more lives down the ages
than perhaps any other infectious illness and has resisted man’s efforts to isolate
and conquer it. A disease once thought to be on the verge of extinction has
rebounded in its most dangerous avatar yet. This chapter describes the culmina-
tion of the hunt for the elusive microbe of tuberculosis and the fascinating
sequence of events that followed.
Keywords
Infection · Tuberculosis · World Health Organization
Abbreviations
BCG Bacillus Calmette-Guerin

DOTS Directly observed treatment
INH Isonicotinylhydrazide
A. Hasan (*)
Department of Respiratory Medicine, Deccan College of Medical Sciences, Hyderabad, India
e-mail: principal@deccancollegeofmedicalsciences.com
# Springer Nature Singapore Pte Ltd. 2019 3

S. E. Hasnain et al. (eds.), Mycobacterium tuberculosis: Molecular Infection Biology,
Pathogenesis, Diagnostics and New Interventions,
https://doi.org/10.1007/978-981-32-9413-4_1
4 A. Hasan
MDR-TB Multidrug-resistant tuberculosis

MTB Mycobacterium tuberculosis
PAS Para-aminosalicylic acid
PPD Purified protein derivative
SCCT Short-course chemotherapy
TB Tuberculosis
WHO World Health Organization
XDR-TB Extensively drug-resistant tuberculosis
Anton van Leeuwenhoek, the Dutch microscopist, had seen protozoa in 1674 and
bacteria in 1676. Since Leeuwenhoek’s specimens were obtained from healthy
individuals, he did not associate microorganisms with disease. The first time tuber-
culosis (TB) was conceptualized as an infection was in 1720. Benjamin Marten in his
A New Theory of Consumption proposed that the etiologic agents of TB were
“wonderfully minute living creatures” which were transmissible by the airborne
route and that “it may be therefore very likely that by an habitual lying in the same
bed with a consumptive patient, constantly eating and drinking with him, or by very
frequently conversing so nearly as to draw in part of the breath he emits from the
lungs, a consumption may be caught by a sound person.” But a century and a half
later, not much progress upon the matter had been made.
Louis Pasteur, the great French researcher—biochemist, microbiologist, and
immunologist par excellence—was instrumental in destroying the Theory of Miasma
and thus paved the way for the incrimination of microbes as the causative agents of
infection. By his time, microscopes were achieving much higher magnifications than
even Leeuwenhoek’s superb lenses (that had been capable of 250 to 300
magnification).
Much later, Ernst Abbe, Professor at the University of Jena, working for Carl
Zeiss took optics to a different level. One of Zeiss’ microscopes found its way into
the hands of a District Medical Officer in the pastoral German district of Wollstein.
Robert Koch, once an army doctor, now settled into the tedium of peacetime
medical practice, became drawn to a mysterious illness rampant among the livestock
of Wollstein—anthrax. At that time, in 1871, after the recently concluded Franco-
German War, some of the bitterness between the two warring nations had spilled
over into medicine itself. Louis Pasteur was already a colossus and a household
name. Yet, despite his successes, he and other notable French scientists had failed to
clarify the life cycle of what was to be called the Bacillus anthracis.
By day, Heinrich Hermann Robert Koch was a typical country doctor. But at
night, with hardly any equipment to speak of (though he did have a microscope
presented to him by his wife on his 29th birthday) and using slivers of wood that
served as lancets, he worked ceaselessly to unravel the life cycle of anthrax.
Professor Ferdinand Cohn was Director of the Botanical Institute at the Univer-
sity of Breslau and widely regarded as the founder of modern bacteriology. Cohn
had described, for the first time, endospores of Bacillus subtilis, a close relation of
what today is known as Bacillus anthracis (Cohn 1872). It was natural that Koch
turned to Cohn. “After many failures,” Koch wrote to him, “I have finally succeeded
1 History of TB: Robert Koch and Beyond 5
in completely elucidating the life cycle of Bacillus anthracis” (Sakula 1982). Cohn,
not a little surprised that a country practitioner using his rudimentary tools was
claiming to have unraveled a mystery that had so far eluded the elite researchers of
France and Germany, promptly summoned Koch, who then, before Cohn and
pathologists Karl Weigert and Julius Cohnheim, proceeded to give a demonstration
of “meticulous methodical research.”
“We thus see that anthrax tissues, regardless of whether they are relatively fresh,
putrefying, dried or many years old can produce anthrax when these substances
contain bacilli capable of developing spores of Bacillus anthracis”—and by stating
this, Koch brought into focus once again the role of germs at the heart of transmissi-
ble disease (Koch 2010).
Cohnheim went on to state “I consider this the greatest discovery in the field of
bacteriology” and prophetically added that “. . .Koch will again astonish and shame
us with still further discoveries” (Wagner 1885).
Unfortunately, Koch’s views found no favor with Rudolph Virchow the highly
influential chief pathologist of Berlin’s Charite Hospital, who also served as leader
of the opposition in the German Parliament. Virchow held that it was an intrinsic
derangement in the function of the body’s cells that caused all disease and dismissed
out of hand the role of “infection” from “external agents.” He considered Koch’s
theory “improbable.”
Koch, disillusioned but determined, returned to Wollstein with a new Carl Zeiss
microscope—with its special new oil immersion lenses crafted by the Ernst Abbe,
the finest lens maker in Germany—that magnified images more than a thousandfold.
In 1879, he published another brilliant paper, “Investigations of the Etiology of
Wound Infections” (Koch 2010), in which he proposed that different types of
infections existed.
When Koch moved to Breslau and then eventually to Berlin—mainly through the
efforts of Cohn and Cohnheim—he took charge of his new laboratory with some of
the most brilliant names to adorn medical textbooks: Friedrich Loeffler (discoverer
of the diphtheria bacillus) and Georg Gaffky (discover of the germ of typhoid and
co-discoverer—with Koch—of the germ of cholera) were assigned to him.
Koch’s new team initially concentrated on refining culture media. Liquid media
had many limitations, not the least of which was that separation of the pathogen from
contaminants was difficult; Koch admitted that it was “truly depressing to attempt
pure cultivation” (Sakula 1982). In 1881, Koch tried garden potatoes, then gelatin,
and finally—on an inspired suggestion of his assistant Walter Hess’ wife and
assistant Fannie—a seaweed extract, agar. Success was immediate. Gratified,
Koch began to use solid media in flat-bottomed glass dishes devised by another of
his illustrious assistants, Julius Richard Petri.
Armed with his novel culture media, Koch finally turned his attention—
secretly—to something close to his heart: tuberculosis!
But despite all the innovations that went behind his culture media and staining
methods (Koch was provided a variety of stains created by yet another brilliant
colleague, Paul Ehrlich), the TB bacillus refused to show itself under the
microscope.
6 A. Hasan
Slightly differential versions of the following story exist: but essentially, it

appears that Koch forgot to discard an old slide of a 10-day-old culture lying near
a warm stove, covered with dye, in the ammonia-heavy air of his laboratory. The
next morning an astonished Ehrlich showed Koch the vivid blue clumps of
mycobacteria (Sakula 1982). The ammonia fumes had permeated into the cell
walls of mycobacteria, and the alkalinity and the heat had carried the methylene
blue dye into the cell. The bacillus lay stark and exposed.
In the spring of 1882, armed with hundreds of slides, test tubes, flasks, culture
plates, human and animal tissue specimens, and the records of nearly
500 experiments, Koch proceeded—not to the Berlin Pathological Society, where
Virchow was influential—but to the Berlin Physiological Society, where, in the
words of Loeffler, he “. . .spoke slowly and haltingly, but what he said was clear,
simple, logically stated—in short, pure unadulterated gold.”
“On the basis of my recent observations” Koch stated, “I consider it as proved that
in all tuberculous conditions of man and animals there exists a characteristic
bacterium which I have designated as the Tubercle Bacillus, which has specific
properties which allow it to be distinguished from all other microorganisms” ([The
etiology of tuberculosis by Dr. Robert Koch. From the Berliner Klinische
Wochenschrift, Volume 19 (1882)] 1982).
The date was 24th of March, a date celebrated today as the World
Tuberculosis Day.
What Robert Koch did on that date was not only to unearth mankind’s most
deadly infectious killer but also to firmly entrench the concept of microbes as agents
of infection, a concept that Pasteur, his old rival, had led credence to. However Koch
was not done with Pasteur yet. In an unprovoked assault on Pasteur at the Fourth
International Congress for Hygiene and Demography in Geneva in September
(Carter 1988), Koch challenged both Pasteur’s research methods and his scientific
temper. Pasteur demanded gratification—the great rivalry between two of
medicine’s colossuses sank to the level of a petty quarrel.
By the end of the decade, with the tenth International Medical Conference in
Berlin approaching, letters personally signed by the Kaiser Wilhelm II went out to
over 6000 delegates, and the pressure on Koch to do something special to showcase
Germany’s medical prowess mounted steadily (Burke 1993).
On the third day of the conference, Koch finally unveiled his much anticipated
vaccine against TB. “I have at last hit upon a substance that has the power of
preventing the growth of the tubercle bacilli not only in the test tube but in the
body of an animal,” read out Koch. But both his emphasis on the word “animal” and
his statement that tuberculin was effective only if the disease were “not too
advanced” were completely drowned by the ensuing pandemonium.
With the world’s expectations raised sky-high, Koch began testing this vaccine in
earnest, beginning with himself. Initially the vaccine seemed to work, and a relieved
Koch pronounced the first human trial successful. Both the British Medical Journal
and the Lancet carried a complete translation of Koch’s paper, and the Lancet
described Koch’s discovery as “glad tidings of great joy” (Dubos 1952).
“The eagerness with which patients seize hold of everything under the name of
remedy borders on madness,” Benjamin Rush had written in 1783 in Thoughts upon
the Causes and Cure of Pulmonary Tuberculosis. The floodgates literally opened.
Patients poured into Berlin to receive the miracle drug. As to its composition, Koch
only said: “As regards the origin and preparation of the remedy, I am unable to make
any statement as my research is not yet concluded; I reserve this for a future
communication. The remedy is a brownish, transparent liquid” (Koch 1890b).
Arthur Conan Doyle, already well known for his detective columns featuring
Sherlock Holmes, undertook a pilgrimage to Berlin to meet the new prophet, Koch.
Though he did not get to meet Koch, he did have a chance to look around and see
what the vaccine could possibly do. Conan Doyle was disappointed in the “cure.”
Yet he foresaw that Koch’s lymph would prove “an admirable aid to diagnosis” and
a “single injection” would help determine if the pathology were “in any way
tubercular.” In all fairness, Koch himself had himself said: “I think I am justified
in saying that the remedy will therefore in the future form an indispensable aid to
diagnosis. By its aid we shall be able to diagnose doubtful cases of phthisis. . .”
(Koch 1890a).
Within the space of a few short months, it was obvious that tuberculin did not
work well. Many patients worsened; several died. The British Medical Journal in an
editorial cautioned, “Already warning voices are being raised in Vienna and in
Berlin in the attempt to stem the tide of misconception which is rolling in a veritable
flood of consumptives to the Prussian capital. . .” (Campbell and Bah-Sow 2006). As
to the formula which Koch still withheld, the editors urged that “. . .our patience is
not to be severely taxed, for it is much to be hoped that Dr Koch. . . .will give at an
early date the promised account of the mode of preparation of his remedy.”
A heavily decorated Koch became the object of censure—and ridicule, even. It
was in 1905—nearly quarter of a century later—that he finally received the award
that long ago should rightfully have been his—the Nobel Prize in Medicine.
Koch’s vaunted vaccine, tuberculin (“old tuberculin”—OT), was after all just a
glycerinated beef-broth culture of heat-killed bacilli. True to the deductions of
Sherlock Holmes’ creator, tuberculin became an important tool for the diagnosis
of tubercular infection—a role it still plays after over 140 years of its discovery.
Austrian theologist and philosopher-turned-pediatrician, Baron Clemens Peter
von Pirquet, found that by superficially scratching the skin through two drops of
diluted OT, a skin reaction could be produced and used to effectively diagnose TB
infection in Viennese children (Pirquet 1909). In 1907, Alfred Wolff-Eisner and
Albert Calmette introduced a wafer of solidified tuberculin between the eyelids of
bovine animals (Paine and Martinaglia 1929). Other methods of inoculating the
subject with tuberculin—some bordering on the repugnant—were explored: Tuber-
culin in various forms was inserted into the nose, rectum, urethra, or vagina.
Fortunately, these methods were soon abandoned. In 1908 in Munich, Ernst Moro
prepared a paste of tuberculin and lanolin (Munchener Medizinische Wochenschrift/
27 February 1912: On transplantation immunity by Georg Schone, Greifswald,
1978) and R Lautier a wad of tuberculin soaked in cotton, the same year, to apply
to the skin (Grozin 1943).
8 A. Hasan
In 1908 Charles Mantoux’s method of injecting tuberculin directly into the

epidermis (Mantoux 1910) replaced Pirquet’s, and with a slight modification by
Felix Mendel, the test took its current form as the popular Mendel-Mantoux—or
simply the “Mantoux”—test.
In 1934, Florence Seibert’s group at the University of Pennsylvania came up with
an improved purified protein derivative (PPD) of tuberculin (Richards et al. 1979),
and the WHO later further improved it by decreasing its adherence to containers by
adding the detergent Tween 80 (Guld et al. 1958). By the late 1990s, a new lot,
PPD-S2, had become the new standard (Villarino et al. 2000).
Today, in spite of its relative unsophistication, the tuberculin skin test—a cocktail
of over 200 mycobacterial antigens—has managed to stand the test of time. In 1997,
Ajit Lalvani and his colleagues at the Imperial College in London developed the
ELISpot assay, an interferon-gamma assay based on a genomic segment unique to
the MTB called the “region of difference-1” (Lalvani et al. 1997).
Yet in spite of everything, the footprint of TB was difficult to see. What was
needed was a way to image the body and see the effect of disease on its tissues.
On November 8, 1895, at the University of Würzburg, 50-year-old Karl Wilhelm
Conrad Roentgen noticed unusual fluorescence in crystals of barium platinocyanide
that were kept in the proximity of a covered Crookes’ tube. Roentgen arrived at the
deduction that a new kind of invisible ray (he called it “X” for want of a better name)
could penetrate and cast images of hidden structures (Tubiana 1996). That very day,
Roentgen photographed a lead pipe and then his wife’s hand. Within just a couple of
years, X-rays had become a new diagnostic tool in medicine. Soon after, Italian
physicist Enrico Salvioni unveiled fluoroscopy, which medicine also “appropriated.”
With these devices, the shadows of tuberculosis were seen everywhere, but
mostly in the lungs. In the 1930s, Brazilian pulmonologist Manuel Dias de Abreu
found a way to take miniature X-rays (Ravindra 2002) and the contraption could be
fitted into mobile units and transported to people’s very doorsteps for community
screening.
In 1901, Röntgen was awarded the first Nobel Prize in Physics, but being
painfully stage-shy, he demurred when asked to give his Nobel acceptance speech.
He never patented his discovery and refused to consider any financial gain from his
invention, donating to his university the money that went with the Nobel.
However, although diagnosis was now a relatively simple, the treatment of TB
was another matter altogether. People relied on rest cures, usually under the sun, for
there was little else.
Pliny the Elder had said, “Sol est remediorum maximum—‘The sun is the best
remedy’ (of all)” (Pliny 1938). Ancient Egyptians and the Babylonians had long
known this; ancient Greeks had Helioses in which they sunbathed (Research 1996)
and the Romans had their Solaria.
Over time, sunbathing, fresh air, and gentle exercise gained popularity as general
measures to treat the ill. Thomas Sydenham the English physician (The “English
Hippocrates”) recommended horse riding (Sydenham and Dewhurst 1966), and
George Bodington, practicing in Coldfield, Sutton, used the combination—minus
the horse—to good effect (Keers 1980). That very year, Otto Walther started his
Nordrach Sanatorium in the Black Forest (McCarthy 2001), and in 1855, Austrian
Arnold Rikli set up another one in Bled, Slovenia, writing no less than seven books
detailing his methods.
Meanwhile, America had already opened its doors to settlers—and to
consumptives in search of a natural cure. Iowa, Minnesota, and Wisconsin in the
late 1840s gained popularity for the restorative qualities of their air and made this
fact public through advertisements targeted at patients with TB.
Just about this time, a freshly graduated doctor’s struggles of over four decades
with TB were just beginning. In 1872, Edward Livingston Trudeau developed
consumption. “I think I knew something of the feelings of the man at the bar who
is told he is to be hanged on a given date,” he wrote, “. . .it seemed to me that the
world had grown suddenly dark. . .my dreams were all shattered now, and only exile
and inevitable death remained” (Trudeau 1916).
Trudeau undertook a long and arduous journey to his beloved Adirondack
mountains where he hoped to die in peace. Instead, he recovered and returned to
his practice at New York City—whereupon he promptly suffered a relapse. He
finally returned to the Adirondacks and made a home there. In 1882, Trudeau read
about the travels of another consumptive doctor, Silesian Hermann Brehmer, who
had regained his health at the foothills of the Himalayas (McCarthy 2001) and gone
on to open a sanatorium-type “Kurhans” in Gorbersdorf. In 1884, in a quaint little
wooden cottage called “Little Red,” Trudeau established the Adirondack Cottage
Sanatorium. By 1950, a total of 100,000 patients could be housed in sanatoria across
the USA.
In 1956, funded by the Indian Council of Medical Research (ICMR) and the
Government of Tamil Nadu in India and supported by the WHO, a landmark study
was performed by Wallace Fox (Watts 2010) at the Medical Research Council at the
newly founded Tuberculosis Chemotherapy Centre at Madras to see if patients could
be effectively treated outside of Sanatoria. Wrote S Radhakrishna: “. . .the outcome
of this trial was critical for the management of tuberculosis in India, as well as other
developing countries with a high disease burden and inadequate resources” (Wallace
2010). After 5 years, the Madras trial proved that the vast majority of patients could
be treated equally effectively at their homes.
In 1888, at about the same time that Trudeau was building “Little Red,” Carlo
Forlanini of Milan described the first successful artificial pneumothorax, intended to
“rest” the lung (Sakula 1983), and in 1911, the invention of the water manometer
made it possible to let in a precise amount of air into the chest—through a special
needle invented by Saugmann. Later, collapse therapy began to be carried out with
Lillington and Pearson’s artificial pneumothorax apparatus under radiologic moni-
toring (Rakovich 2010). Over two-thirds of all patients admitted to sanatoria with all
but the most minor forms of TB had collapse therapy performed on them. Andrew
Banyai soon introduced a related procedure—the pneumoperitoneum—to push up
patients’ diaphragms and further immobilize the lung. In some patients, the phrenic
nerve was divided—either in isolation or in combination with pneumoperitoneum—
to achieve the same end.
10 A. Hasan
The introduction of air into the pleural cavity was frequently impossible on
account of the pleural adhesion to the thoracic wall that many patients had.
Ferdinand Sauerbruch, the German thoracic surgeon—author of the first textbook
on thoracic surgery—devised extrapleural thoracoplasty in which excision of the
upper ribs allowed the cavity-cavity-containing lung apices to collapse inwards. In
1904, Sauerbruch performed surgery with the patient’s torso enclosed within his
pressure chamber (Cherian et al. 2001) which was capable of ventilating the patient
by generating both negative and positive pressure.
In 1910, Swede Hans Christian Jacobaeus, while yet an intern, adapted a cysto-
scope to serve as a laparoscope, and later as a thoracoscope in 1922, describing
40 cases of pleural adhesiolysis (Jacobaeus 1922).
In 1947, to make the after-effects of thoracic surgery less disabling, Nagaishi and
Wilson began inserting hollow spheres of polymethylmethacrylate—“lucite
spheres”—into the apical pleural space. The procedure went by the unedifying
name of “plombage” (Vigneswaran 1995). Heavy oil was sometimes used,
“oleothorax,” but it sometimes produced unpleasant inflammatory reactions and on
occasion tracked into the bronchial tree with disastrous consequences.
In spite of everything, the 5-year mortality was close to a 100%. What was needed
was an antibiotic that killed mycobacteriae.
The wonder drug penicillin was completely ineffective against the mycobacteriae
whose highly complex cell walls appeared to have no vulnerability. Aspirin for some
reason had quite the opposite effect: it stimulated a burst of activity. Bernheim
remarked on this in a letter to Danish biochemist Jorgen Lehmann who was working
on perfecting an anticoagulant, dicoumarol (Lehmann 1964). Later, Lehmann used
the same strategy that he had used to construct dicoumarol. He swapped an amino
group from the “ortho” to the “para” position, and the new variant of aspirin, para-
aminosalicylic acid (PAS), showed effects opposite to that aspirin had—it inhibited
bacterial synthesis.
In spite of disappointments with their earlier anti-TB drug ventures, Ferrosan, a
Danish pharma company, decided to hedge on PAS. Chief chemist Karl Rosdahl
churned out a meagre 13 grams for Lehmann, which Lehman routed through to
Gylfe Vallentin, Head of Renstrom’s Sanatorium (Lehmann 1964).
On October 1944, a full month before Selman Waksman used streptomycin in the
USA, 24-year-old Sigrid was commenced on PAS—she recovered. In April 1947,
the Swedish National Board for Tuberculosis began a major trial on PAS, which was
later, on the evidence of a historic trial by the British Medical Council (Treatment of
pulmonary tuberculosis with streptomycin and para-aminosalicylic acid; a Medical
Research Council investigation 1950), incorporated into a combination regimen for
TB. Over the next 20 years, the annual production of PAS rocketed to 3000 tons.
Meanwhile in America, Ukraine-born Selman Waksman, a widely regarded
expert on soil microbes, was convinced that “The cure of tuberculosis must come
not from enzymes, but from antibiotics.” Yet, though his team had already processed
over 10,000 specimens, it was to no avail.
In 1943, the tide turned. Twenty-three-year-old Albert Schatz joined Waksman’s

team. Less than 2 months later, one of Waksman’s students, Doris Jones, presented
the team with organisms cultured from the croup of a chicken from which Schatz—
and another recent addition to the team, undergraduate student, Elizabeth Bugie—
succeeded in isolating streptomycin. It was a while before the drug was put to use.
“The discovery had consequently been made, but was not discovered by the
discoverers themselves!” observed Birath in the Scandinavian Journal of Respira-
tory Disease.
At the Mayo Clinic, William H Feldman and physician Corwin Hinshaw finally
undertook to test streptomycin. After several successful experiments on guinea pigs
(Lehmann 1964), on November 20, 1944, the first round of injections was given to a
seriously ill 21-year-old in Mineral Springs Sanatorium in Cannon Falls, Minnesota.
Patricia weighing all of 75 pounds took a series of injections. She survived, going on
to get married and have three children.
Although patients treated with either streptomycin or PAS appeared to do well at
first, they soon relapsed. Yet, when combined, the drugs went on to become the
keystone for anti-TB therapy. Waksman went on to win the Nobel Prize for the year
1952; Albert Schatz, who had always considered himself the true discoverer of
streptomycin, and Jorgen Lehmann—arguably the discoverer of the first effective
anti-tubercular agent—did not.
Then, in 1952, INH or, properly, isonicotinic-acid hydrazide was simultaneously
discovered by three separate teams at three separate labs—teams led by Harry Yale
at the Squibb Institute in New Brunswick, by Robert Schnitzer at Hoffmann-La
Roche in Nutley, and by Gerhard Domagk in Germany. Domagk had discovered the
anti-tubercular drug thiacetazone shortly before (McDermott 1969).
Then, Lepetit Research Labs in Milan brought forth the best drug for TB yet.
Franco Parenti, holidaying in France in 1959, returned home with unusual gifts for
his colleague Piero Sensei—clods of earth from a pine forest near Nice. From the
Nocardia mediterranei he found within, Sensei extracted a new TB drug (Sensi et al.
1959) which he called rifomycin (after Rififi—The Struggle—a movie Parenti had
fancied) (Dubos 1952).
In 1968, rifampin was launched in Europe and 6 years later in the USA. Ninety-
five percent of all patients who took a rifampicin-INH combination-based regimen
were pronounced cured, and anti-tubercular therapy was shortened to 9 months. In
1961 came ethambutol from Lederle Labs; and when pyrazinamide arrived, the anti-
tubercular armory was complete; the extent of any TB regimen that initially
incorporated four drugs was further reduced to a “mere” 6 months.
Meanwhile, radiology was undergoing a sea change. In 1973 the first CT scanners
were unveiled at EMI and almost immediately thereafter at the Tufts University at
Massachusetts by Allen McLeod Cormack, working independently. By 1970, Paul
Lauterbur, a chemical engineer, was working on the first magnetic resonance
imaging (MRI) scanner at the University of Illinois. Peter Mansfield a physicist at
the University of Nottingham, UK, improved upon the techniques.
12 A. Hasan
Although both Lauterbur and Mansfield got the 2003 Nobel Prize (Macchia et al.
2007), Raymond Damadian, who had constructed an MRI body scanner in 1969
(Harold 1972)—and had performed what was probably the first human MRI scan in
1977—did not (Damadian 1971).
By 1914, the world’s first maritime SONAR machine had become fully func-
tional in the USA largely through the efforts of Canadian Reginald Fessenden (Woo)
and soon was adapted to medicine’s needs.
With both diagnostic radiology and TB therapeutics up to speed, all that the world
needed now was a preventive vaccine.
The twentieth century saw a violent beginning—World War I. Under the almost
continuous bombing that went on overhead, at Lille, veterinarian and immunologist
Camille Guérin along with the Director of the Pasteur Institute at Lille, Léon Charles
Albert Calmette, began to wage a daily battle against the tubercle bacillus. In 1919,
after incredible 11 years and 231 subcultures, they succeeded in weakening the
Mycobacterium enough to make a vaccine of it (Weir et al. 2008).
In 1921, newborns at the Charité Hospital in Paris were successfully injected the
vaccine of Bacillus Calmette-Guérin—now called the BCG. Over the next 9 years—
between 1924 and 1925—the vaccine was exported to over 30 nations, and in 1928 it
gained the seal of approval of the Health Committee of the League of Nations. But
then, disaster struck! In 1930, in the German town of Lübeck, nearly all of the
249 infants who were administered the BCG vaccine developed TB, and 76 died
over the following months. Overnight, almost, the remedy had turned into a poison.
It was eventually found that there was nothing wrong with the vaccine itself: a
batch of BCG had become inadvertently contaminated with a virulent strain of
tubercle bacillus, but by then, a devastated Calmette had passed on. BCG made its
expected resurgence, and post-World War II—between 1945 and 1948—eight
million babies in Eastern Europe received the vaccine.
Although the safety of the vaccine was proven beyond doubt, questions about its
efficacy remained. Complexities in the vaccination programs run by different
countries, racial and regional factors of the local population, and the technical skills
of the operators—and, indeed, the differences in the vaccine strains used—impacted
the efficacy of the vaccine (Weir et al. 2008).
In 1968, an ICMR-WHO-US Public Health Services-funded joint committee was
appointed to study the vaccine, and a vaccine incorporating the Pasteur and Danish
strains was administered to 360,000 people in the district of Chingleput 40 km from
Chennai in India. Twelve and a half years later, the reports showed that protection
rate in children was just 17%; that in adults was zero. A follow-up paper from the
ICMR concluded: “. . .BCG offers no protection against adult type bacillary tuber-
culosis. Consequently, BCG cannot be expected to reduce the transmission of
tuberculosis” (Tripathy 1987).
Nevertheless, in 1974, the BCG was included in the Expanded Program on
Immunization, and by 2003, in most areas the use of BCG grew to achieve 95%
coverage. Today BCG has been made mandatory in 64 countries and officially
recommended in almost twice as many more (Colditz et al. 1994), and over four
billion individuals have so far received the vaccine (Nuttall and Eley 2011).
In 1998, the Sanger Centre in Cambridge, Britain, and Paris’ Pasteur Institute in
France cracked the genetic code for the old H37Rv strain of tubercle bacillus, and
then the genome of highly virulent CDC1551 (the “Oshkosh” strain) was mapped at
the TIGR (Fine 1989).
By the 1980s, in the developed nations at least, TB seemed to be in decline. By
the mid-1980s, this decline was no longer as manifest, and in some developed
countries, the graph of new cases had begun to climb again.
A new disease had arrived.
The HIV epidemic pushed up the epidemiological curve of TB, and by December
1997, 11.7 million individuals had perished, with 16,000 new infections occurring
each day. By the turn of the millennium, as the developed world inevitably came to
terms with the HIV and gained a measure of control on it, the disease spiraled out of
control in Asia and Africa. By 2015—the year the WHO had predicted would usher
in a catastrophe—the situation had actually improved (UNAIDS 2015). Still, nearly
40 million people globally were living with HIV worldwide (74% of them in
sub-Saharan Africa), but new HIV infections had fallen by 35% and the global
response to HIV had saved millions from contracting the disease—even
tuberculosis-related deaths had fallen by 32% since 2004 (UNAIDS 2015).
Footnote: Down the ages, a double armed heraldic cross had served as the
emblem of the kings of Sumeria, flown upon the pennants of Joan of Arc, fueled the
hermetic revival of René d’Anjou, and was revered as the Patriarchal Cross of
Jerusalem. Later, it became the mark of the infamous Godfrey, Duke of Lorraine.
By 1967, four frontline drugs were available for treating TB: INH, pyrazinamide,
ethambutol, and rifampicin. Wallace Fox and other researchers in British Medical
Research Council units in East Africa, India, Hong Kong, and Singapore combined
these drugs together into an effective regimen that shortened the treatment to
6 months. Short-course chemotherapy (SCCT) became the standard treatment
for TB.
On the basis of a paper from the Tuberculosis Chemotherapy Centre, Madras, that
showed the effectiveness of a twice-weekly administration of TB drugs, it was
realized that treatment could now be dispensed under direct supervision. This new
biweekly regimen came to be called the DOTS (World Health Organization 1994;
Morse 1996).
Thus, after peaking in 2004, the incidence rates of TB began to decline, and by
2014, all six of WHO’s regions had achieved the modest the Millennium Develop-
ment Goal. TB was still at large, killing 1.5 million persons, and new unsettling
words were increasingly being used—multidrug-resistant TB (MDR-TB) and exten-
sively drug-resistant TB (XDR-TB). That year in 2014, 190,000 individuals died of
MDR-TB.
According to WHO figures, nearly 10% of all MDR-TB have already
metamorphosed into the deadlier XDR already—in over 100 countries (World
Health Organization 2015b). The problem was that the world has run out of drugs
to treat these new forms of TB (Ventola 2015).
14 A. Hasan
Two new anti-TB drugs—bedaquiline and delamanid—have finally broken

through the deadlock, but drug resistance to these virtually unused drugs is already
being reported (Bloemberg et al. 2015).
As well, improving health technology has begun to drive earlier TB diagnosis
(Sulaiman et al. 2013), and “rapid culture” tests—MODS, Griess, MGIT, thin-layer
agar, colorimetric assays, and others—are competing for preference, along with the
now well-accepted CBNAAT and line-probe assays (Medecins Sans Frontiers
2015).
Despite everything, given the new 2030 targets, TB control has been lagging
nearly 150 years behind schedule (Hasan 2017). The WHO’s End-TB Strategy
2016–2035—a costed, scalable blueprint for a world free of TB, zero deaths and
zero disease and suffering due to TB—reflects the world’s growing desperation to be
done with a disease that for too long has remained unchallenged as mankind’s most
destructive infection. The new goals are by far the most ambitious yet (World Health
Organization 2015a). As things stand today, things are far from perfect (Hasan
2017). TB accounts for a quarter of all avoidable deaths in developing nations.
Robert Koch’s in his Nobel acceptance speech had said, “The fight has been
ignited fully and the enthusiasm for this goal is so broad that I am not afraid that it
will cease again...If we continue to work in such a powerful way, victory will be
achieved.”
Now, if ever, is the moment of truth.
Footnote: The 2025 milestones for the End-TB Strategy are a 75% reduction in
TB deaths (compared with 2015), a 50% reduction in TB incidence rate (less than
55 TB cases per 100,000 population), and no affected families facing catastrophic
costs. The 2035 milestones are 95% reduction in TB deaths (compared with 2015),
90% reduction in TB incidence rate (less than 10 TB cases per 100,000 population),
and no affected families facing catastrophic costs due to TB.
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www.who.int/tb/publications/global_report/en
Tuberculosis as an Underlying Etiological
Factor for Other Human Respiratory 2
Diseases
Ronan F. O’Toole
Abstract
Tuberculosis (TB) does not occur in isolation from other human illnesses. There
are multiple examples where TB combines with one of more comorbidities to
amplify its prevalence. Noncommunicable diseases such as diabetes, or lifestyle
behaviors including smoking and alcohol misuse, place people at a greater risk of
presenting with active TB. But the epidemiological associations between TB and
other human conditions are not confined to increasing susceptibility to TB disease.
TB, in itself, is an underlying risk factor for the development of downstream
respiratory illnesses later in life. This indicates that injury to the host resulted
from an episode of TB persists beyond successful eradication of Mycobacterium
tuberculosis infection by antimicrobial drug therapy. In this chapter, the specific
role of TB in promoting other lung diseases is examined. In particular, TB during
childhood increases the risk of development of progressive and poorly reversible
airway diseases that include bronchiectasis and chronic obstructive pulmonary
disease. It is apparent from the literature that prevention of TB disease offers a
potential pathway for reducing the global burden of downstream chronic lung
diseases.
Keywords
Tuberculosis · Mycobacterium tuberculosis · Bronchiectasis · Chronic obstructive
pulmonary disease
R. F. O’Toole (*)
School of Molecular Sciences, College of Science, Health and Engineering, La Trobe University,
Melbourne, VIC, Australia
Department of Clinical Microbiology, School of Medicine, Trinity College Dublin, Dublin, Ireland
School of Medicine, College of Health and Medicine, University of Tasmania, Hobart, Tasmania,
Australia
e-mail: otooler3@tcd.ie

https://doi.org/10.1007/978-981-32-9413-4_2
18 R. F. O’Toole
Abbreviations
AIDS acquired immunodeficiency syndrome

BOLD Burden of Obstructive Lung Disease
CI confidence interval
COPD chronic obstructive pulmonary disease
FEV1 forced expired volume of air in the first second of expiration
FVC forced vital capacity
HIV human immunodeficiency virus
MDR multidrug resistant
OR odds ratio
TB tuberculosis
UI uncertainty interval
UK United Kingdom
2.1 Background
Tuberculosis (TB) is a major cause of infectious disease mortality worldwide, killing

approximately 1.67 million people in 2016 alone (World Health Organization 2017).
There were an estimated 10.4 million new cases of the disease in 2016 corresponding
to an average annual incidence rate of approximately 140 per 100,000 persons
globally (World Health Organization 2017). This included 490,000 cases that were
multidrug resistant (MDR) and a further 110,000 cases that were resistant to
rifampicin but susceptible to isoniazid in 2016. The involvement of HIV/AIDS in
increasing the risk of TB development is well established (Chaisson and Martinson
2008; Geldmacher et al. 2008, 2010; Hwang et al. 2013; Swaminathan et al. 2000).
Of the new cases of TB in 2016, 1.03 million occurred in HIV-positive individuals
(World Health Organization 2017).
Noncommunicable diseases also contribute to the burden of TB. A systematic
review by Jeon and Murray of 13 observational studies, comprising 17,698 TB
patients, found that diabetes was associated with an approximate threefold height-
ened risk of developing active TB (relative risk of 3.11 [95% CI, 2.27–4.26]) (Jeon
and Murray 2008). Diabetes is also a risk factor for adverse treatment outcomes in
TB patients (Lonnroth et al. 2014).
A population-based study of 115,867 patients in Sweden, aged 40 years and
with a hospital discharge diagnosis of chronic obstructive pulmonary disease
(COPD), found that the relative risk of TB development in COPD patients was
threefold higher than in controls (hazard ratio, HR, of 3.0 [95% confidence interval,
2.4–4.0]) (Inghammar et al. 2010). Furthermore, COPD patients who developed
active TB exhibited a twofold greater risk of death from all causes in their first year
post TB diagnosis with respect to general population controls with TB (odds ratio,
2 Tuberculosis as an Underlying Etiological Factor for Other Human. . . 19
Bronchiectasis
Compression of the bronchi by enlarged

hilar and interlobar lymph nodes during
childhood TB can result in obstruction,
atelectasis and dilatation, and increases
the risk of bronchiectasis
Tuberculosis Intrinsic obstructive and stenotic lesions

of the bronchi during adult TB can lead to
chronic atelectasis and retractile fibrosis
and increase the risk of bronchiectasis
Global Burden
10.4 million new cases in 2016 est.
1.67 million deaths in 2016 est. COPD
Incidence rate of 140 / 100,000 est. Pulmonary TB can lead to a remodelling

of the lung architecture manifested as
extensive fibrosis, cavitation,
bronchostenosis or parenchymal lung
destruction
This results in a deterioration in lung

function as measured by FEV1 (forced
expired volume of air in the first second
of expiration) and FVC (forced vital
capacity) and increases the risk of
development of COPD
Fig. 2.1 Relationship between tuberculosis and the subsequent increased risk of development of
airway diseases bronchiectasis and chronic obstructive pulmonary disease (COPD)
OR, of 2.2 [95% CI, 1.2–4.1]) (Inghammar et al. 2010). A study conducted on
23,594 COPD patients in Taiwan, and 47,188 non-COPD control subjects matched
for age and gender, also identified COPD as an independent risk factor for TB
development with a hazard ratio of 2.468 [95% CI, 2.205–2.762] (Lee et al. 2013)
(Fig. 2.1).
Tobacco smoking has been well established as a dominant causative factor for the
development of COPD (Lokke et al. 2006; Lundback et al. 2003). The percentage of
COPD mortality attributable to smoking has been estimated to be in the region of
54% for men aged 30–69 years and 52% for men older than 70 years of age (Eisner
et al. 2010). Smoking is also a risk factor for the development of TB. Following a
meta-analysis of 24 studies, Bates and colleagues reported that smoking represents a
significant risk factor for Mycobacterium tuberculosis infection as well as TB
disease with relative risks estimated at 1.73 [95% CI, 1.46–2.04] and 2.33 [95%
CI, 2.15–3.28], respectively (Bates et al. 2007). Other studies have corroborated the
link between smoking and TB (Slama et al. 2007; Lin et al. 2007). Alcohol misuse is
also a risk factor for TB (Lonnroth et al. 2008; Fok et al. 2008) as it is for ischemic
heart disease (Laatikainen et al. 2003; Klatsky 2009).
Therefore, it is evident that TB shares risk factors with a range of human illnesses.
As we will see in the proceeding sections, the relationship between TB and other
20 R. F. O’Toole
conditions extends to its own role as an underlying risk factor for long-term airflow
limitation of the respiratory system. The objective of this chapter is to review the
epidemiological data that link TB and subsequent airway disease.
2.2 TB and Bronchiectasis
One of the first chronic airway diseases to be associated with post-tuberculosis was
bronchiectasis which is defined as an “irreversible localized or diffuse dilatation,
usually resulting from chronic infection, proximal airway obstruction, or congenital
bronchial abnormality” (Cantin et al. 2009). In conjunction with enlargement of the
bronchi is an impaired ability to maintain effective mucociliary clearance, an
important innate defense mechanism in the bronchial tubes. This immune deficit
permits microbes and other particles to accumulate in the mucus layer leading to
inflammation and further damage of the airways.
In terms of the burden of bronchiectasis, global prevalence rates are not available;
however, they have been estimated in a number of countries. The prevalence of
bronchiectasis in the USA was previously calculated at 25 per 100,000 in the general
population and 272 per 100,000 in persons aged 75 years and above (O’Donnell
2008). But the growing use of high-resolution computed tomography has
contributed to an increase in the number of people diagnosed with bronchiectasis
in the USA. Using healthcare claims data from 2009 to 2013, Weycker and
co-workers determined the prevalence of bronchiectasis in the general US popula-
tion to be 139 cases per 100,000 based on narrow case-finding criteria (Weycker
et al. 2017). Age variability was evident with a prevalence rate of 7 per 100,000
observed in persons aged from 18 to 34 years and 812 per 100,000 in persons aged
75 years and above (Weycker et al. 2017). The authors concluded that the burden of
bronchiectasis was higher than previously reported. In Germany, the total number of
individuals with bronchiectasis in 2013 was estimated to be 53,807, corresponding
to a prevalence rate of 67 [95% CI, 64–69] per 100,000 general population
(Ringshausen et al. 2015).
Granger is attributed with recognizing in 1878 that bronchiectasis occurred as a
consequence of TB (Grancher 1878). Jones and Cournand referred in 1933 to an
enlargement of lymph nodes in primary tuberculosis as a possible causative factor in
the development of bronchiectasis (Jones and Cournand 1933). In 1935, Wallgren
cited the frequency of bronchiectasis in children after primary tuberculosis
(Wallgren 1935). The etiological link between tuberculosis and bronchiectasis was
established in a number of follow-up studies. These included the work by Valledor
and colleagues published in 1952 (Valledor and Navarrete 1952). They examined
250 cases of primary tuberculosis in children using serial bronchoscopy and
bronchography over a period of 15 years. They reported bronchiectasis of varying
degrees in approximately 70% of cases (Valledor and Navarrete 1952). They
attributed the cause of bronchiectasis to compression of the bronchi by enlarged
hilar and interlobar lymph nodes during childhood TB leading to obstruction,
atelectasis, and dilatation. They also examined TB in adults and reported that
bronchiectasis in adult TB results from intrinsic obstructive and stenotic lesions of

the bronchi culminating in chronic atelectasis and retractile fibrosis (Valledor and
Navarrete 1952). Based on their work, they concluded that “bronchiectasis is a
common sequel of chronic pulmonary tuberculosis, generally affecting the upper
lobes.”
While TB has been a major source of bronchiectasis, the introduction of anti-
tubercular chemotherapeutics and other interventions to control the disease is
believed to have reduced the incidence of TB-related bronchiectasis (Bilton and
Jones 2011). This is evident in studies performed in low TB incidence countries such
as the UK. In 2000, Shoemark and co-workers reported that among 165 patients with
confirmed bronchiectasis at the Royal Brompton Hospital, London, only 2 patients
had bronchiectasis associated with post-TB (Shoemark et al. 2007). Similarly, in a
study conducted on 150 bronchiectasis patients at Papworth Hospital, Cambridge,
UK, only 3 patients had a history of TB (Pasteur et al. 2000).
Studies have also been conducted in countries which have a high incidence of TB
to examine the role of TB in contributing to the burden of bronchiectasis. In India,
which had an estimated 2.79 million cases of TB in 2016, the highest number of TB
cases in the world (World Health Organization 2017), one study involving a small
number of bronchiectasis patients found that pulmonary TB was the most common
etiology (10/19 or 52.63% of patients) (Nag et al. 2015). In earlier work by Sahoo
and colleagues, it was reported that among 100 pulmonary cases of TB with
detectable residual lung lesions in chest x-rays after completion of chemotherapy,
62 had bronchiectasis (Sahoo et al. 1988). In China, which had an estimated 895,000
cases of TB in 2016 (World Health Organization 2017), a study of non-cystic
fibrosis bronchiectasis patients found that pulmonary TB was a major cause of
bronchiectasis (2175/6977 or 31.17% of patients) ahead of pertussis and other
bacterial infections (Xu et al. 2013). Therefore, it is apparent that reducing the
burden of TB could assist in decreasing the prevalence of chronic lung diseases
such as bronchiectasis, particularly in high TB incidence countries.
2.3 TB and COPD
Chronic obstructive pulmonary disease (COPD) refers to chronic airflow obstruction

that is progressive and poorly reversible and includes chronic bronchitis and emphy-
sema (Pauwels et al. 2001). The disease originates with structural damage to the
small airways and neighboring alveoli and progresses over a number of years
culminating in a reduction of elastic recoil and an increase in airway resistance
(Petty 2006). These pathologies cause defects in lung function, measurable with
spirometry, that become more pronounced with advancing age (Petty 2006). In terms
of burden, COPD is emerging as the third highest cause of human mortality globally
after heart disease and stroke (World Health Organization 2014). In 2015, 3.2
million people [95% uncertainty interval, UI, 3.1–3.3 million] died from COPD
worldwide (Collaborators 2017). The World Health Organization (WHO) has
estimated that over 65 million people are afflicted with moderate to severe COPD
22 R. F. O’Toole
(World Health Organization 2015). Recent data have found that the prevalence of
COPD grew by 44.2% between 1990 and 2015 to 174.5 million individuals [95%
UI, 160.2–189.0 million] (Collaborators 2017).
Tuberculosis is increasingly been recognized as a cause of obstructive pulmonary
disease. A meta-analysis published in 2015 found a significant correlation between a
past history of TB and the presence of COPD in individuals aged 40 years or more
when adjusted for known COPD risk factors such as cigarette smoking and age
(Byrne et al. 2015). Importantly, when the national annual TB incidence rate
exceeded 100 cases per 100,000 population, the odds ratio (OR) of COPD develop-
ment in patients who had a previous history of TB was more than 3 times that of
patients with history of TB. For example, the adjusted OR for adults in the
Philippines was 6.31 [95% CI, 2.67–15.0], and for males and females in
South Africa were 4.9 [95% CI, 2.6–9.1] and 6.6 [95% CI, 3.7–11.7], respectively
(Byrne et al. 2015).
In their review of nonsmoking-related COPD, Salvi and Barnes reported that
almost half of fully treated TB patients later presented with obstructive airway
disease when examined during follow-up (Salvi and Barnes 2009). In a study
conducted on miners in South Africa, it was found that both FEV1 (forced expired
volume of air in the first second of expiration) and FVC (forced vital capacity) were
reduced in parallel with an increasing number of episodes of TB (Jordan et al. 2010).
In addition, the Korean National Health and Nutrition Examination Survey
2008–2012 reported that a previous history of pulmonary TB (OR of 2.314 [95%
CI, 1.922–2.785]) and an inactive pulmonary TB lesion (OR of 2.300 [95% CI,
1.606–3.294]) represented predictors for subsequent obstructive airway disease [25].
In an analysis of patients hospitalized with COPD exacerbation (n ¼ 598), Yakar and
colleagues reported that COPD diagnosis and death was observed 5 years earlier in
patients who had a past history of TB (Yakar et al. 2017). Mattila and co-workers,
from a study of 6701 adults in Finland, determined the adjusted OR for airway
obstruction ranged from 2.21 [95% CI, 1.52–3.21] in patients who had a TB scar
recorded by a radiologist to 4.59 [95% CI, 2.86–7.37] in patients who had a known
history of TB disease (Mattila et al. 2017).
A large, international, population-based Burden of Obstructive Lung Disease
(BOLD) study examined the association of airflow obstruction and spirometric
restriction with a previous history of TB. Examining a study population of 14,050
participants in 18 different countries, the study established a self-reported history of
TB to be significantly associated with airflow obstruction (adjusted OR of 2.51 [95%
CI, 1.83–3.42]) and also spirometric restriction (adjusted OR of 2.13 [95% CI,
1.42–3.19]) (Amaral et al. 2015). Based on the findings of the BOLD study, the
authors concluded that a history of tuberculosis “should be considered as a poten-
tially important cause of obstructive disease and low lung function, particularly
where tuberculosis is common” (Amaral et al. 2015). Therefore, multiple strands
of evidence now support the role that TB plays in increasing the likelihood that
patients may go on to develop COPD in later life.
A number of studies have examined the specific aspects of TB pathology that
affect lung structure and function. Pulmonary TB can result in extensive fibrosis,
cavitation, traction bronchiectasis, bronchostenosis, or parenchymal lung destruction

(Jordan et al. 2010; Dheda et al. 2005). In a South African-based study, Wilcox and
Ferguson determined that in subjects with a previous history of TB treatment who
were undergoing pulmonary function assessment, airway obstruction was prevalent
in 68% of cases (Willcox and Ferguson 1989). They concluded that “treated
pulmonary tuberculosis is a cause of significant chronic obstructive airways disease”
(Willcox and Ferguson 1989). While antimicrobial chemotherapy may lead to an
improvement in lung function in patients with pulmonary TB, Plit et al. (Plit et al.
1998) reported the presence of residual airflow limitation and a restrictive pattern in a
sizeable proportion of patients, i.e., 28% and 24%, respectively. In a study conducted
in India, 78% of obliterative bronchiolitis cases were identified as being post-TB
(Gothi et al. 2007). Chronic deficits in lung function as estimated by spirometry were
found by Hnizdo and colleagues to correlate with the number of TB episodes in
which functional decreases of 18.4%, 27.1%, and 35.2% were associated with one,
two, and three or more episodes of TB, respectively (Hnizdo et al. 2000). Therefore,
airflow obstruction and a loss of pulmonary function can manifest in patients who
have completed their TB treatment (Ehrlich et al. 2011). Pasipanodya and colleagues
have shown that years lived with disability due to TB extend well beyond the acute
TB disease due to long-term pulmonary impairment (Pasipanodya et al. 2010).
Furthermore, significantly reduced lung function, as measured by FVC
(43.58 16.03% versus 72.06 14.95% predicted) and FEV1 (33.08 15.64%
versus 66.13 19.87% predicted), was detected in patients who had the multidrug-
resistant (MDR) form of TB with respect to patients who had non-MDR-TB
(Di Naso 2011). It is therefore apparent that episodes of active TB cause structural
and functional changes that heighten the risk of subsequent progression to COPD
even after successful completion of TB treatment.
2.4 Conclusions and Future Perspectives
Epidemiological associations are emerging between communicable and

noncommunicable respiratory diseases. TB in itself worsens the condition of the
lungs such that they become more prone to chronic airway illnesses including
bronchiectasis and chronic obstructive pulmonary disease. A previous history of
TB increases significantly the propensity for subsequent bronchiectasis or COPD
development even after the patient has successfully adhered to and completed their
prescribed TB chemotherapy. These findings heighten the concern that the immense
global incidence of TB and MDR-TB could be sustaining an underdiagnosed level of
chronic airway disease, particularly, in high TB burden countries. The WHO
predicts that mortalities due to COPD are destined to increase in the decades to
come (Mathers and Loncar 2006); therefore, reducing the burden of TB may be an
important strategy in reversing that trend. Further research is now required to define
the level of contribution that TB makes to the incidence of bronchiectasis and
COPD. In addition, investigations are required to establish the specific mechanistic
relationships that exist between TB and chronic airway diseases at the cellular and
24 R. F. O’Toole
molecular levels. An improved level of understanding of the interactions between

TB and noncommunicable lung illnesses may translate into better interventions for
the prevention, detection, and management of respiratory disease.
Funding Not applicable.
Competing Interests The author declares that he has no financial or other conflicts of interest.
Ethical Approval Not required.
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Host Factors in Tuberculosis
3
Ruxana T. Sadikot
Abstract
Tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB) continues to
remain one of the most deadly infectious diseases worldwide especially in the
developing countries. The lack of new drugs for TB, vaccine availability, with
increasing global antimicrobial resistance, has prompted more research into
defining host factors and developing immunomodulatory strategies. Some of
the known risk factors that increase the susceptibility to TB include immunocom-
promised states such as human immunodeficiency virus (HIV) infection, smok-
ing, socioeconomic deprivation, malnutrition, diabetes mellitus, air pollution,
occupational exposures, and alcohol consumption. In this chapter we review the
available information about host factors that help understand susceptibility to
TB. Defining the host factors is critical to develop host-directed therapies that
may enhance host defenses and perhaps reduce tissue damage from chronic TB
infections.
Keywords
Mycobacterium tuberculosis · Human immunodeficiency virus · Diabetes
mellitus · Tumor necrosis factor · Interferon
Abbreviations
AIDS Acquired immunodeficiency syndrome

DM Diabetes mellitus
EPTP Extrapulmonary tuberculosis
R. T. Sadikot (*)
Section of Pulmonary, Critical Care Medicine, Emory University, Atlanta, GA, USA
e-mail: ruxana.sadikot@emory.edu

https://doi.org/10.1007/978-981-32-9413-4_3
28 R. T. Sadikot
HIV Human immunodeficiency virus

IFN Interferon
MBL2 Mannose-binding lectin gene
TB Tuberculosis
TNF Tumor necrosis factor
3.1 Introduction
Tuberculosis is the predominant infectious disease in mankind and the ninth leading
cause of death globally, ranking on top of HIV/AIDS. Mycobacterium tuberculosis,
the infectious organism, is carried by an estimated 2000 million people worldwide,
and data from WHO show that it takes around 1.5 million lives every year (WHO
2017; Kyu et al. 2018). A variety of newer circulating strains are being identified,
which have geographic variation, which are increasing the complexity of the disease.
With advances in the diagnostic techniques and molecular genotyping, these
variations are becoming apparent and being extensively studied. Similarly, a better
understanding of host factors that increase the propensity for infection needs to be
better defined.
It is increasingly recognized that there are several host factors that increase the
propensity to develop TB such as demographics, genetics, socioeconomic variations,
smoking, alcoholism (Simou et al. 2018), and immune status of the individual
(Narasimhan et al. 2013). Some of the host factors that are related to genetics
which increase predilection for TB occurrence and progression are being better
defined with advances in human genome (Chapman and Hill 2012; Khor and
Hibberd 2012). Ecological factors that are associated with distribution and variation
of the disease constitute important targets for larger interventions to reduce TB
infections. In this chapter host factors that have been associated with TB occurrence
are discussed.
3.2 Gender Differences
In general, most studies have reported a significantly higher occurrence of TB in men

than in women. By employing epidemiological surveys, Horton et al. have identified
that this disease affects men population more than the women population, with a
ratio close to 2 as reported by worldwide case notification rates (Horton et al. 2016).
This gender-biased frequency of TB represents disease epidemiological patterns, but
the reasons for this biasness are still unclear and need to be better understood. This
phenomenon is quite well known and has been reported widely in different settings
and geographic areas over the last century (Dolin et al. 1998; Diwan and Thorson
3 Host Factors in Tuberculosis 29
1999; Begum et al. 2001; Nhamoyebonde and Leslie 2014; Ting et al. 2014). It is
becoming evident that the reasons for the observed gender bias may be far more
complex and have been controversial in literature. It is likely that both gender- and
sex-related factors may contribute to higher tuberculosis rates in men.
Biological differences may explain some of the differences in sex-biased rates of
infection and disease. These include factors such as differences in immunity and
variations in exposure to MTB associated with different social mixing patterns. Of
note there is also a difference in health-seeking pattern among women especially in
developing countries where resources are limited and cultural inhibitions of women.
These are some of the epidemiological data that have been associated with poorer
detection of disease among females (Bothamley et al. 1998). However, there is a
scarcity of conclusive epidemiological evidence to support some of these theories.
However, gender inequalities in accessing healthcare should be investigated more
rigorously as this is a potentially correctable factor and may have significant impact
on disease prevalence and spread as women in developing world also care for
children who may get infected. Another important factor that may contribute to
these differences includes lack of women reporting in passive-case finding contexts.
Additionally, other factors such as misuse of alcohol and excessive smoking, which
are generally more prevalent in men, may also contribute to the increased incidence
of TB in men. Two separate reports have proposed that smoking is one of the key
parameters for the observed differences in the incidence of TB in males and females
(Maurya et al. 2002; Gajalakshmi et al. 2013).
While socioeconomic and cultural factors could be a cause in creating barriers to
accessing healthcare leading to undernotification in women, in recent years,
emerging data have also shown that biological mechanisms may contribute to the
differences between men and women in susceptibility to TB infection. Interestingly,
studies have shown that women have a better ability to clear pathogens, which has
been linked to sex steroid-induced immunomodulation. A study by Nhamoyebonde
showed that there is a difference in the effects of sex hormones such as the influence
of androgens on gut microbiota, which may provoke host susceptibility to TB
(Nhamoyebonde and Leslie 2014; Khan et al. 2016). Other studies have shown
that sex hormones have different effects on immune cell function. For example,
estrogen induces and testosterone represses T-helper cells and can impact macro-
phage stimulation and humoral immune responses, which play an important role in
tuberculosis immune responses (Grossman 1985). Despite of these observations, the
scientific evidences to define the immune differences are scant. Further studies are
needed to investigate these biological gender differences to better define how sex
differences predispose to TB.
3.2.1 Socioeconomic Factors
In developing countries, socioeconomic risk factors may play a key determinant role
in the development and progression of TB disease and may impact the recurrence
and development of resistance (Mohidem et al. 2018). Several studies have identified
these determinants and include lack of social protection, low income, lack of
30 R. T. Sadikot
availability of healthcare services, poor living conditions such as ventilation of

residences, a high average number of inhabitants per household, and overall lack
of hygienic environment (Souza et al. 2008; Hargreaves et al. 2011; Sales et al.
2018). Household income, educational level, and occupation leading to hazardous
exposures have been evaluated more extensively in studies as socioeconomic
factors. Data from several studies point toward the impact of poverty on treatment
compliance as a particularly important factor. Other socioeconomic factors such as
poor nutrition and hygiene are a result of poor living conditions. Malnutrition can
particularly impact the innate and adaptive responses and may result in increased
predilection for the disease and occurrence of TB (Sinha et al. 2018a, b).
In a recent study from Indonesia, Renggannis Wardani et al. showed that educa-
tion, housing density index, and internal house transmission are the foremost
parameters related to latent TB. Thus, TB control program should be integrated
with improvement in education, hygiene, and a more rigorous examination of
internal house contacts (Renggannis and Wahono 2018). Furthermore, it should be
emphasized that these programs should be supported by public and health
institutions. Such measures will likely advance TB control programs, particularly
in low- and middle-income nations where with high-socioeconomic disparity is
observed.
Worldwide, prisoners are also considered as one of the vulnerable groups to
developing TB. Screening for incarcerated individuals should be more rigorous and
systematic, as they may also be responsible for infecting other inmates. Understand-
ing of socioeconomic differences has several practical implications for TB control
programs. There is a requirement to strengthen screening for precise subgroups
within the prisoners, in individuals with deprived socioeconomic class and
comorbidities, along with consideration of probable role of systematic screening
for latent TB infection. These findings reinforce the need for a community-centered
approach for control of TB in areas of high endemicity and the need to deploy
financial resources to these sectors which will have a broader impact on TB control
programs.
3.3 Smoking and Alcohol
Smoking has been found to be a modifiable risk factor for TB infection and disease
although the evidence is not yet considered to be conclusive (Kolappan and Gopi
2002). In general, smoking impairs innate and adaptive immune response (Mehta
et al. 2008). A case-control study of smoking and mortality from India provides
some evidence of this link although the study was limited to examining mortality
among males as low smoking frequency among females. This study reported that
smoking is attributing to the 61% of urban adult male mortality due to tuberculosis.
The study also showed that smoking is responsible for around four times more
deaths caused by tuberculosis as compared to nonsmokers. These limited data
provide some evidence although larger epidemiologic and scientific studies to
confirm the effect on smoking and TB are much needed.
A meta-analysis of 24 studies by Bates et al. showed that smoking is a risk factor

for TB infection and disease (Bates et al. 2007). However, whether smoking
contributes to increased mortality risk in persons who already have active TB is
not established. The data from study by Bates et al. suggest that tuberculosis control
policies should incorporate tobacco control including chewing and smoking cessa-
tion as a preventive intervention. A pooled analysis further reported hazard ratios
were increased by threefold for developing TB in relation to alcohol consumption
during follow-up. Study using exposure-response analysis reported that alcohol
intake (10–20 g every day) leads to 12% increase in TB risk (Bates et al. 2007).
Studies have revealed that smoking is connected with impairment of immune
function and may be a risk factor to development of infections (Mehta et al. 2008).
Together, these data suggest that smoking cessation should be considered as an
integral component of TB control programs.
3.4 Diabetes Mellitus
Many studies suggest an increased risk of pulmonary tuberculosis among those with
diabetes mellitus (DM) which poses a significant independent risk for the develop-
ment of active tuberculosis. DM can also worsen disease outcome and complicate
treatment. Tuberculosis and diabetes mellitus are both common and independently
have immense public health significance globally. The association and consequences
of the two diseases have been well-established in countries where the incidence of
both the diseases is high. DM might impact the disease presentation and response to
treatment. This is particularly true for patients who have a poor glycemic control.
Recent studies suggest that this may be related to alterations in both innate and
adaptive immune responses related to poor glycemic control. It is also interesting
that diabetic patients who are smear-positive for TB have a higher possibility of
failure for smear conversion compared to patients without DM.
Recent studies have recognized that TB can stimulate glucose intolerance and
exacerbate dysglycemia in patients with DM. However, evidence on whether the
association is bidirectional is yet to be established. In a recent study from the United
Kingdom, Pearson et al. (2018) found that DM risk was substantially raised among
individuals with a history of TB disease. This finding has implications for follow-up
and screening for patients with TB, who may be at high risk of developing DM or
related complications. In countries such as India where the incidence of TB is high
and that of DM is increasing, this association has major health implications.
There is lack of clear evidence of whether TB-DM comorbidity is co-related with
increased risk of developing multidrug-resistant tuberculosis (MDR-TB). In a recent
meta-analysis, Tegegne et al. reviewed 24 observational studies from 15 different
countries. Their analysis discovered that DM is highly associated with MDR-TB and
DM can significantly enhance the probabilities of developing MDR-TB. Therefore,
TB-DM patients must be provided with a more robust TB treatment and follow-up
strategies. Additionally, efforts to control DM can have a considerable beneficial
effect on TB consequences, specifically in patients who have MDR-TB (Tegegne
32 R. T. Sadikot
et al. 2018). Other immunodeficiencies such as HIV can exacerbate the impact of
DM and TB. By employing a retrospective database, in a community healthcare
setting in KwaZulu-Natal Province of South Africa, Sinha et al. showed that DM and
HIV synergistically increased the odds of TB symptoms (Sinha et al. 2018a, b).
However, further studies are needed to define the impact of glycemic control on
development of MDR-TB. With increased understanding with metabolomics, more
scientific studies are needed to better define the host defense effects of hyperglyce-
mia and TB propensity.
3.5 Genetics
The genes that impact immune responses are diverse as infectious diseases exert
significant selective genetic pressure (Abel et al. 2017). Thus, the impact of host
genetic variability on the susceptibility to exogenous pathogens is well recognized.
Studies related to genomic analysis have revealed connection between
polymorphisms in multiple histocompatibility complex and human leukocyte anti-
gen genes that induces susceptibility to TB infection and disease. Modern studies
that have employed genome-wide association approach have yielded several novel
findings to define the susceptibility to TB. Some of these have been confirmed in
experimental settings. The evidences also suggested the significant role of human
genetic polymorphisms in susceptibility to latent TB infection and advancement of
active infection such as mannose-binding lectin gene.
The host-pathogen relationship in TB has been described as sympatric (Fenner
et al. 2013). This would mean that the pathogen and host may share some common
ancestral geographic origin. When patients are infected with ana strain that
originates from a different geographic origin than the patient, then the host may
have a risk for greater lung pathology and impairment (Pasipandoya et al. 2013).
Similarly, there is also evidence for associations between susceptibility to TB and
human leukocyte antigen which depends on the strain of infection (Toyo-Oka et al.
2017). There is data that suggests an association of mannose-binding lectin gene
(MBL2) polymorphisms with susceptibility to tuberculosis; however the data is not
conclusive. Cao et al. performed a meta-analysis of 22 case-control studies to assess
the effect of MBL2 polymorphisms and the risk of developing tuberculosis. They
found that MBL2 rs1800451 polymorphism is linked with lower TB risk in the
general population, and A/O, rs7096206, rs1800450, and rs1800451 were connected
with the risk for some explicit ethnic groups (Cao et al. 2018). These data highlight
the significance of genetic factors that can predispose to TB. As the genomic
approaches to identify single nucleotide polymorphisms become more sophisticated,
these associations will be better defined. This will help identify patients who are at
high risk of developing TB or may be protected from TB infection and disease.
3.6 Immunocompromised Patients
It is well recognized that patients who have immunosuppressed states such as HIV,
patients with solid or bone marrow transplant, and those who are on biologics are
predisposed to developing TB. The prevalence of TB among people living with
HIV/AIDS (PLWHA) is high. The incidence of both pulmonary and extrapulmonary
TB is increased in PLWHA. Many studies from different countries have estimated
the burden of combined HIV and TB. The prevalence is particularly high in
sub-Saharan Africa and India.
Recent data suggest that the incidence of extrapulmonary TB is also considerably
high. Mohammed et al. analyzed 31 studies with a total population of 28,659 from
1990 to 2017. They found that the prevalence of extrapulmonary TB (EPTB) among
PLWHA ranged from 6.4% (Mohammed and Assefa 2008). Studies have shown that
TB- and HIV-coinfected patients have a high mortality. In particular, a high mortal-
ity rate was observed during the first 3 months in those patients who did not take
ART (Tshitenge et al. 2018). Thus, it is recommended that patients with HIV
infection should be screened for TB early in their care, to minimize delays in
diagnosis and treatment. This would allow for immediate treatment for both active
and latent infection. This is particularly important in developing countries in Africa
and Asia where there is a rise in HIV infection. It is also important to test TB cases
for HIV. These rigorous screening programs will contribute to reduced morbidity
and mortality among people living with AIDS. To reduce the burden of TB, the
WHO’s policy on collaborative TB/HIV activities recommend several measures for
HIV-infected individuals. These include intensified case finding, isoniazid preven-
tive therapy, infection control, and antiretroviral therapy for patients with HIV.
3.7 Immune Therapeutics and Susceptibility to TB
The recent advent of monoclonal antibodies and soluble receptors that target various
molecules such as tumor necrosis factor (TNF)-α has modernized the therapeutic
approach for many autoimmune and inflammatory diseases. The connection between
anti-TNF therapy and reactivation of latent TB or de novo TB is well recognized.
Due to the pleiotropic functions performed by TNF-alpha, blockade of TNF-α may
intensify the incidence of serious infections such as TB. Physiological
TNF-mediated signaling could be impaired by anti-TNF therapy and may lead to
reactivation and dissemination of latent TB infection (Garziera et al. 2017). The
maintenance of the granuloma is important for the containment of TB. Immune
signaling through TNF and interferon IFN-γ is compulsory for host defenses against
M.tb infections. TNF is important for multiple processes such as macrophage
activation and recruitment of other immune cells including natural killer cells and
lymphocytes, which either allows the formation of granuloma or kills the bacterium.
TNF helps in the maintenance of the granuloma in patients with latent TB infection.
TNF-mediated immune-inflammatory responses are attenuated by anti-TNF therapy,
causing disruption of well-formed granulomas. This can cause a release of viable
34 R. T. Sadikot
mycobacteria which in turn can lead to reactivation of TB (Cantini et al. 2018).

Hence, it is recommended that careful screening is essential for patients who are to
commence anti-TNF therapy.
3.8 Summary
Despite the significant efforts made by the World Health Organization (WHO) and
partners to control TB, it is still a major public health issue. Recognition of risk
factors that predispose to TB can help identify at-risk patients. Specialized
interventions should be implemented to tackle the risk factors associated with this
dreadful disease specifically in a high-risk population. Socioeconomic factors play
an important role in the progression of TB disease and will need to be considered in
TB control programs; these include smoking cessation and misuse of drugs and
alcohol. Similarly, screening programs for patients with immunocompromised states
especially those with HIV and those who receive biologics are recommended. Other
correctable factors such as alcoholism and malnutrition may also play a critical role
in TB control. Early diagnosis and rigorous treatment can prevent emergence of
resistance and limit functional impairment of lungs. Studies to establish the gender
differences are also much needed. Further research into the role of genetic factors
and indoor and outdoor physical environments to better understand the association
between the physical environment and the social environment with TB infection are
much needed.
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Extrapulmonary Tuberculosis
4
Surendra K. Sharma and Alladi Mohan
Abstract
Extra-pulmonary tuberculosis (EPTB) refers to an isolated occurrence of tuber-
culosis (TB) at body sites other than the lung. The advent of human immunodefi-
ciency virus (HIV) and acquired immunodeficiency syndrome (AIDS) pandemic
and widespread use of immunosuppressive drugs and biologicals have changed
the epidemiology of EPTB. In immunocompetent individuals, EPTB constitutes
15–20% of all TB; in immunosuppressed individuals, EPTB accounts for more
than 50% of the cases. Because of paucibacillary nature of the disease and
limited accessibility to the disease site, it is difficult to establish the diagnosis.
However, advent of newer imaging methods like 18F-labelled 2-deoxy-D-
glucose positron emission tomography (18FDG-PET)-computed tomography
(CT) and PET-magnetic resonance imaging (MRI) has helped in better
localisation and defining the extent of EPTB. With the availability of new
molecular tools such as nucleic acid amplification tests (NAAT) and line
probe assays (LPA), liquid culture method, the rapid diagnosis of EPTB, drug
and drug susceptibility testing (DST)-guided treatment are possible. However,
EPTB still remains a diagnostic and therapeutic challenge. Further, its treatment
is cumbersome since endpoints are not well defined. For better adherence and
S. K. Sharma (*)
Departments of General Medicine & Respiratory Medicine, JNMC, Datta Meghe Institute of
Medical Sciences (DMIMS), Wardha, Maharashtra, India
Department of Internal Medicine (WHO Collaborating Centre for Research & Training in
Tuberculosis, Centre of Excellence for EPTB, MoH & FW, GoI), All India Institute of Medical
Sciences, New Delhi, Delhi, India
e-mail: sksharma@aiims.ac.in
A. Mohan
Department of Medicine, Sri Venkateswara Institute of Medical Sciences, Tirupati, India

https://doi.org/10.1007/978-981-32-9413-4_4
38 S. K. Sharma and A. Mohan
successful treatment outcome, active TB Drug-Safety Monitoring and Manage-

ment (aDSM) is recommended.
Keywords
Tuberculosis, extrapulmonary · Diagnosis · Cartridge-based nucleic acid
amplification tests · Treatment · Human immunodeficiency virus · Multidrug-
resistant tuberculosis
Abbreviations
ATS American Thoracic Society

CBNAAT Cartridge-based nucleic acid amplification tests
CDC Centers for Disease Control and Prevention
CT Computed tomography
DR-TB Drug-resistant TB
E Ethambutol
EPTB Extrapulmonary TB
18 18
FDG-PET-CT F labelled 2-deoxy-D-glucose positron emission
tomography
H Isoniazid
IDSA Infectious Disease Society of America
INDEX TB guidelines Indian Extra-pulmonary TB guidelines
IRIS Immune reconstitution inflammatory syndrome
MDR-TB Multidrug-resistant TB
MRI Magnetic resonance imaging
R Rifampicin
RR-TB Rifampicin-resistant TB
TB Tuberculosis
TBM TB meningitis
Z Pyrazinamide
4.1 Introduction
Tuberculosis (TB) is one of the top 10 causes of death among adults in the world. As
per the Global TB Report 2018 (Global tuberculosis report 2018), in 2017, ten
million people developed TB disease; TB caused an estimated 1.3 million deaths
among human immunodeficiency virus (HIV)-seronegative people and an additional
300,000 deaths among HIV-seropositive individuals (Global tuberculosis report
2018). Drug-resistant TB (DR-TB) continues to remain a major public health
4 Extrapulmonary Tuberculosis 39
problem world over. Multidrug-resistant TB (MDR-TB, defined as isolates of

Mycobacterium tuberculosis (M. tb) resistant to isoniazid and rifampicin, with or
without resistance to other anti-TB drugs)/rifampicin-resistant TB (RR-TB) has been
documented in 3.6% of new TB cases and 17% of previously treated cases in 2017
(Global tuberculosis report 2018). Almost one-fourth of the world’s population
(23%, 1.7 billion) are estimated to have a latent TB infection (Global tuberculosis
report 2018). With use of invasive sampling and widespread use of molecular
diagnostic tests, extrapulmonary TB (EPTB) is increasingly being diagnosed and
notified. In this chapter, we have attempted to provide a critical overview regarding
the clinical detection, diagnosis and treatment of the more commonly encountered
forms of EPTB.
4.2 Terminology
Pulmonary TB is the most common presentation of TB. The term “EPTB” refers to
specific presence of TB in different body parts other than the lung. When an
extrapulmonary focus is obvious in a patient with pulmonary TB, such patients are
categorised as pulmonary TB (Sharma and Mohan 2004, 2013).
By this categorisation, intrathoracic mediastinal and/or hilar lymph node TB or
TB pleural effusion, without radiographic abnormalities in the lungs, is categorised
as EPTB; miliary TB is categorised as pulmonary TB.
The term “disseminated TB” refers to active TB disease characterised by
coexisting participation of at least two noncontiguous body sites or evidence of
M. tuberculosis in the blood or bone marrow. “Miliary TB” is a kind of disseminated
TB as a consequence of a massive haematogenous dissemination of tubercle bacilli
which results in minute discrete foci typically the size of millet seeds (1–2 mm)
distributed consistently in the lungs and the other viscera (Sharma et al. 2005, 2012).
4.3 Epidemiology
In the pre-HIV era and in immunocompetent adults, EPTB constituted about

15–20% of all cases of TB (Sharma and Mohan 2004). In HIV-seropositive people
with late HIV infection, the risk of TB increases as immunosuppression progresses.
In immunosuppressed individuals, EPTB accounts for more than 50% of all cases
of TB (Fig. 4.1) (Sharma and Mohan 2004). As per the Global TB Report 2018
(Global tuberculosis report 2018), EPTB constituted 14% of the 6.4 million
incident TB cases (Table 4.1). EPTB can develop during primary or postprimary
(reactivation, reinfection) TB. Peripheral lymph node TB and TB pleural effusion
are two common forms of EPTB; DTB and MTB are more frequently encountered
in HIV-seropositive and immunosuppressed individuals (Sharma and Mohan 2004,
2013).
Fig. 4.1 Distribution of tuberculosis cases by anatomical site in immunocompetent (a) and
immunosuppressed (b) adults
PTB pulmonary tuberculosis, EPTB extrapulmonary tuberculosis, GUTB genitourinary tuberculo-
sis, DTB disseminated tuberculosis, MTB miliary tuberculosis, TBM tuberculosis meningitis, ABD
abdominal tuberculosis, LNTB lymph node tuberculosis
Reproduced with permission from Sharma and Mohan (2004)
Table 4.1 Proportion of WHO region Total cases notified EPTBa (%)
EPTB cases from among
Africa 1,375,550 16
notified TB cases in 2017
The Americas 228,943 15
Eastern Mediterranean 523,494 24
Europe 220,832 16
Southeast Asia 2,826,486 15
Western Pacific 1,350,202 08
Global 6,444,278 14
Data source: Global tuberculosis report (2018)
EPTB extrapulmonary TB, TB tuberculosis, WHO World Health
Organization
a
Includes new and relapse EPTB cases for which the “treatment
history is unknown”. It excludes cases that have been reregistered
as treatment after failure, as treatment after lost to follow-up or as
other previously treated (whose outcome after the most recent course
of treatment is unknown or undocumented)
4.4 Clinical Presentation
4.4.1 Constitutional Symptoms
Patients with EPTB often (though not always) manifest general constitutional
symptoms such as fever, anorexia, weight loss, malaise and fatigue. For example,
fever is seldom present in peripheral lymph node TB; but, when the disease is
positioned at an obscure site, EPTB may present with pyrexia of unknown origin
(PUO), and this may be the only diagnostic hint in these patients (Sharma and
Mohan 2004).
4.4.2 Symptoms and Signs Related to Site of Involvement
In addition to constitutional symptoms, patients with EPTB manifest symptoms of

the organ system involved, and these are described under the respective anatomical
sites (Sharma and Mohan 2004).
4.4.3 Lymph Node TB
Peripheral lymph nodes are most often affected site of EPTB. Lymph node TB is
considered to be the local manifestation of a systemic disease and may develop at
the time of primary infection or may occur in later stages of life as postprimary
TB. Lymph node TB often affects children and young adults; a female preference
has been reported in several studies. Peripheral lymph node TB has been classi-
fied into (i) stage 1, enlarged, firm mobile discrete nodes; (ii) stage 2, enlarged
nodes fixed to surrounding tissue (periadenitis); (iii) stage 3, central softening

(abscess formation); (iv) stage 4, collar-stud abscess; and (v) stage 5, sinus tract
formation (Jones 1962).
Patients usually present with slowly enlarging lymphadenopathy and may other-
wise remain asymptomatic. Though not common, fever and other constitutional
symptoms may sometimes be evident in some patients. Cervical lymph nodes are
usually affected followed by axillary and inguinal lymph nodes. Multifocal involve-
ment, intrathoracic and intra-abdominal lymphadenopathy and associated pulmo-
nary disease are more common in HIV-seropositive and immunosuppressed
individuals (Sharma and Mohan 2004, 2013).
Physical examination findings are dependent on the stage of the disease. The
enlarged lymph nodes could be of variable size, are typically firm and may be
discrete or matted. Cystic consistency may be seen if necrosis and abscess formation
have occurred. The lymph nodes are not tender in the absence of secondary bacterial
infection. Lymph node abscess can burst causing chronic non-healing sinus and
ulcer formation. TB sinuses have thin, bluish, undermined edges with scanty watery
discharge (Sharma and Mohan 2004, 2013).
4.4.4 Uncommon Manifestations
Uncommon manifestations include dysphagia, oesophago-mediastinal/tracheo-

oesophageal fistula, chylothorax, cardiac tamponade (mediastinal lymph node
involvement), chylous ascites and chyluria (abdominal lymphadenopathy). Biliary
obstruction due to enlarged lymph nodes can cause obstructive jaundice (Sharma
and Mohan 2004).
4.4.5 Pleural Effusion and Empyema Thoracis
TB pleural effusion develops when a small subpleural focus ruptures in the pleural
space and leads to the interaction between the tubercle bacilli or their particular
components prompting a delayed-type hypersensitivity reaction. Other mechanisms
such as rupture of a cavity having caseous material in the pleural space, rupture of
caseous paratracheal lymph nodes, paravertebral abscess or osteomyelitis of the ribs
can also result in empyema thoracis.
TB pleural effusion typically presents as an acute illness. The duration of
symptoms may vary from a few days to few weeks. Pleuritic chest pain, dyspnoea
and nonproductive cough are the most common symptoms. Fever is often present,
with few exceptions. Rarely the onset could be subacute, with only mild chest pain,
cough, low-grade pyrexia, anorexia and weight loss. Patients with TB empyema
present with chest pain, dyspnoea, productive cough, fever and symptoms of TB
toxaemia. Anaemia and hypoproteinaemia are often present.
Physical examination may reveal digital clubbing (empyema), findings sugges-
tive of pleural effusion and intercostal tenderness. Sometimes, TB empyema may
exist as a chest wall mass with an expansile cough impulse (empyema necessitatis) or
a draining sinus tract.
4.4.6 Abdominal TB
The term “abdominal TB” in clinical usage encompasses TB of the gastrointestinal

tract, peritoneum, omentum, mesentery and its nodes and other solid intra-
abdominal organs such as the liver, biliary tract, spleen and pancreas (Sharma and
Mohan 2004).
4.4.6.1 Gastrointestinal TB
Gastrointestinal TB can develop primarily within the intestinal tract or secondary to
a focus elsewhere in the body. The most common site of involvement in gastroin-
testinal TB includes the ileum, ileocecal valve and caecum. Other sites of involve-
ment include the colon, jejunum, stomach, duodenum and oesophagus.
Intestinal TB can develop at any age but is commonly seen in persons aged
between 20 and 40 years; both genders are equally affected. Constitutional
symptoms like fever, weakness, weight loss and night sweats are often present.
Common presenting symptoms include colicky abdominal pain, distension, nausea,
vomiting, constipation and rarely gastrointestinal bleeding (ulcero-constrictive dis-
ease). Diarrhoea may be seen in one-fourth to one-third of the patients. Patients with
intestinal TB may also present with recurrent episodes of intestinal colic, partial or
complete intestinal obstruction. Sometimes, patients may present with weight loss
and pyrexia of unknown origin or features of malabsorption syndrome (Sharma and
Mohan 2004).
4.4.7 Peritoneal TB
Peritoneal TB presents as a subacute disease and presents in three forms, namely,

ascitic (wet type), encysted (loculated) and dry (plastic) forms. Abdominal pain that
is often diffuse and non-localising is the most common presenting symptom.
Abdominal distension is usually present except in the dry form. Tenderness on
palpation is present in almost half of the patients. The classical “doughy” abdomen
is seen in dry or plastic type of peritoneal TB (Sharma and Mohan 2004).
4.4.7.1 Hepatobiliary, Pancreatic and Splenic Tuberculosis

Hepatobiliary and pancreatic TB are rare and are encountered in immunosuppressed
individuals and in miliary TB. The clinical presentation is non-specific. Constitu-
tional symptoms, such as low-grade fever, anorexia, malaise, weight loss and night
sweats, are often present. Other manifestations include malaena, pancreatic mass or
abscess or obstructive jaundice. Clinical presentation of pancreatic TB may mimic
acute or chronic pancreatitis or malignancy (Sharma and Mohan 2004).
While splenomegaly is common in patients with disseminated/miliary TB,

isolated splenic TB is rare in immunocompetent individuals. Splenic TB presents
as fever of unknown origin; imaging may reveal splenic abscess which may be
multiple in immunosuppressed individuals (Sharma and Mohan 2004, 2013).
4.4.8 Neurological TB
Neurological TB is five times more frequent in immunosuppressed individuals

compared with immunocompetent individuals.
4.4.8.1 TB Meningitis
TB meningitis (TBM) accounts for 7–80% of cases and is secondary to TB else-
where in the body (Sharma and Mohan 2004). In developing countries, TBM is a
childhood disease but is frequently being seen in adults as well. Onset is subacute
over 2–6 weeks; nevertheless, acute onset has also been observed. The prodromal
phase (2–3 weeks) is characterised by a history of vague ill-health, irritability, apathy,
anorexia and behavioural changes. With the onset of meningitis, fever, headache and
vomiting, focal neurological deficits and features of raised intracranial tension may
become evident. Focal or generalised seizures and cranial nerve palsies (the sixth
cranial nerve involvement being the most common) are often seen. Complete or
partial loss of vision is a main complication of TBM. Progressive deterioration in the
level of consciousness, pupillary abnormalities and pyramidal signs may indicate
increasing hydrocephalus and tentorial herniation. Deep coma and decerebrate or
decorticate posturing ensues. Untreated, death usually occurs in 5–8 weeks. Uncom-
mon presentations include acute meningitic syndrome, progressive dementia, status
epilepticus, psychosis, stroke syndrome, locked-in state, trigeminal neuralgia, infan-
tile spasm and movement disorders (Sharma and Mohan 2004, 2013).
Patients with TBM are divided into three clinical stages: stage 1, patients are
conscious and oriented with or without signs of meningeal irritation, but there is no
focal neurological deficit; stage 2, altered sensorium or focal deficits are evident; and
stage 3, coma and dense deficits are present. Clinical staging is helpful in optimising
therapy and to predict the prognosis (Kennedy and Fallon 1979).
4.4.8.2 Other Forms of Neurological TB

Other forms of neurological TB include intracranial tuberculomas and single, small,
enhancing brain lesions presenting with epilepsy and arachnoiditis.
4.4.9 TB and the Heart
Pericardial TB is the most common manifestation of TB of the heart. Pericardial

involvement usually results from direct extension of infection from adjacent medi-
astinal lymph nodes or through lymphohaematogenous route from a focus else-
where. The following stages of TB pericarditis have been described: (i) dry stage, (ii)
effusive stage, (iii) absorptive stage and (iv) constrictive stage. However, the disease
may progress sequentially from first to fourth stage or could be present as any stages.
TB pericarditis occurs most frequently in the third to fifth decade of life. The
onset is insidious onset and patients present with fever, malaise and weakness and
vague chest pain. Dyspnoea, cough and weight loss are commonly present. Pericardial
TB can present as acute pericarditis, cardiac tamponade, pericardial effusion, effusive
constrictive pericarditis or chronic constrictive pericarditis. Pulsus paradoxus, raised
jugular venous pressure, ascites and dependant oedema are present; pericardial rub
may be evident (Sharma and Mohan 2004).
Myocardial TB is increasingly being recognised. Patients with myocardial TB
present with idiopathic ventricular tachycardia or unexplained heart failure (Mohan
A et al. 2015).
4.4.10 Bone and Joint TB
Skeletal TB results from haematogenous dissemination and affects almost all bones.
TB of the spine and hip joint is common; other sites include the knee, foot bones,
elbow, hand bones and rarely shoulder joint. Granular and exudative (caseous) forms
are described; and one form may predominate.
Spinal TB is the most frequently encountered form of skeletal TB. Mostly the
affected individuals are under the age of 30 years. Constitutional symptoms, such as
fever, weakness, anorexia, weight loss and night sweats, are usually present and may
manifest before the symptoms related to the spine manifest. Lower thoracic and
lumbar vertebrae are the mostly affected locations of spinal TB followed by middle
thoracic and cervical vertebrae. Classically, two adjoining vertebrae are affected but
several vertebrae may be involved and skip lesions also occur.
The disease process begins commonly in the cancellous area of vertebral body in
epiphyseal location and rarely in central or anterior area of vertebral body. It spreads
and destroys the epiphyseal cortex, the intervertebral disc and the adjacent vertebrae.
The vertebral body becomes soft and gets easily compressed resulting in either
wedging or total collapse. The exudate penetrates the ligaments and follows the
path of least resistance to distant sites from the original bony lesion resulting in cold
abscess formation. The most serious complication of spinal TB is Pott’s paraplegia
and may develop in almost one-third of the patients with spinal TB. Early-onset
(during the active phase of disease) and late-onset (several years after the disease has
become quiescent) paraplegia have been described.
Clinical presentation of TB of the hip and knee joints depends on the clinico-
pathological stage and is characterised by definite patterns of clinical deformity.
Pain, arthritis and “night cries” may be evident. TB osteomyelitis may resemble
chronic osteomyelitis due to other causes (Sharma and Mohan 2004).
4.4.11 Genitourinary TB
Genitourinary TB develops due to haematogenous dissemination from an active site

of TB; descending spread of infection, inflammation and scarring occur. Patients
present with dysuria, haematuria which may be painless, flank pain, renal mass,
sterile pyuria and recurrent urinary tract infection. Rarely, an acute presentation
mimicking pyelonephritis has also been described. Other uncommon presentations
include non-healing wounds, sinuses or fistulae and haemospermia among others
(Sharma and Mohan 2004).
4.4.12 Female Genital TB
Female genital TB develops secondary to TB elsewhere in the body; haematogenous

or lymphatic spread is the most common method of spread. Female genital TB is an
important cause of infertility. Patients may also present with chronic lower abdomi-
nal or pelvic pain or menstrual abnormalities. Symptoms of TB toxaemia may not be
apparent. Physical examination may be unremarkable (Sharma and Mohan 2004).
4.4.13 Cutaneous TB
Several clinical types of cutaneous TB have been reported. These include lupus
vulgaris, scrofuloderma and tuberculosis verrucosa cutis; other lesions seen are
tuberculids (lichen scrofulosorum, papulonecrotic tuberculid, erythema induratum
and erythema nodosum). Miliary TB of the skin and TB chancre have also been
described. Further, localised and generalised skin complications due to bacille
Calmette-Guerin (BCG) vaccination have also been described (Sharma and
Mohan 2004).
4.4.14 Ocular Tuberculosis
Ocular TB usually develops as a result of haematogenous dissemination. TB can

affect all the parts of the eye; choroid is the most commonly affected site. Conjunc-
tival TB and lupus vulgaris are the common manifestations of primary TB, while
tuberculids and phlyctenulosis occur in postprimary TB (Sharma and Mohan 2004).
4.4.15 Tuberculosis in Otorhinolaryngology
Before the advent of anti-TB treatment, patients with active pulmonary TB often
developed laryngeal, otological, nasal and paranasal sinus TB and deteriorated
progressively. These forms have become rare with the availability of effective
anti-TB treatment and are rarely seen presently in immunocompetent persons

(Sharma and Mohan 2004).
4.4.16 TB of the Breast
TB of the breast can occur as primary disease or can be secondary to TB elsewhere in

the body. Secondary involvement of the breast is more common and occurs as a
result of lymphatic or haematogenous spread or by contiguous spread from the ribs,
pleural space. Clinical presentation is atypical, and often, histopathological evidence
suggests the diagnosis (Sharma and Mohan 2004).
4.4.17 Disseminated/Miliary Tuberculosis
Dissemination can occur during primary infection or later during reactivation of

latent infection. Clinical manifestations of disseminated/miliary TB are non-specific.
Clinical presentation is often subacute. Fever and inanition are relatively common.
Cough and dyspnoea are frequently present. Chills and rigors can be present.
Physical examination may reveal peripheral lymphadenopathy and cutaneous
involvement. Choroidal tubercles (bilateral pale greyish white oblong patches), if
present, can be a valuable diagnostic hint to miliary TB. Signs of hepatic involve-
ment (icterus, hepatomegaly) and neurological involvement (meningitis or
tuberculomas) are common. Clinically significant cardiac or renal involvement is
rare. Overt adrenal insufficiency may be evident at initial presentation or may
develop during treatment. Rarely, acute respiratory distress syndrome and acute
kidney injury can develop in patients with miliary TB (Sharma and Mohan 2013;
Sharma et al. 2012).
4.5 Diagnosis
In patients clinically suspected to have EPTB, attempt should be made to procure

sputum; most accessible tissue/body fluids for histopathological, cytopathological,
microbiological and molecular diagnosis. With the availability of ultrasonography,
computed tomography (CT) and magnetic resonance imaging (MRI), 18F labelled
2-deoxy-D-glucose positron emission tomography (18FDG-PET)-CT, PET-MRI,
bronchoscopy, mediastinoscopy, thoracoscopy, upper gastrointestinal endoscopy,
colonoscopy, laparoscopy, cystoscopy, colposcopy, among others, procurement of
tissue and fine needle aspiration and cytopathology (FNAC) under visual/image
guidance, precise anatomical localisation of the lesions of EPTB antemortem and
establishing definitive diagnosis has become possible (Sharma and Mohan 2004;
Sharma and Mohan 2013).
Case definitions for EPTB are shown in Table 4.2 (Sharma et al. 2017). With
availability of “universal drug-susceptibility testing (DST)” at the time of initial
Table 4.2 Case definitions for EPTB

Case Definition
Presumptive case A patient with symptoms and signs of EPTB who needs to be
investigated
Bacteriologically A patient who has a microbiological diagnosis of EPTB, based on
confirmed case positive microscopy, culture or a validated PCR-based test
Clinically diagnosed A patient with negative microbiological tests for TB (microscopy,
case culture and validated PCR-based tests), but with strong clinical
suspicion and other evidence of EPTB, such as compatible imaging
findings, histological findings, ancillary diagnostic tests or response to
anti-TB treatment
Source: Sharma SK et al. (2017)
TB tuberculosis, EPTB extrapulmonary tuberculosis, PCR polymerase chain reaction
presentation (Technical and Operational Guidelines for TB Control in India 2016;

Guidelines on programmatic management of drug-resistant tuberculosis in India
2017 2017) and molecular diagnostic methods for diagnosis of EPTB (Sharma
et al. 2014, 2017; Kohli et al. 2018), especially cartridge-based nucleic acid amplifi-
cation tests (CBNAAT) (Fig. 4.2), rapid diagnosis of EPTB is possible.
The utility of Xpert MTB/RIF in the diagnosis of some forms of EPTB as
recommended in the recently published evidence-based Indian Extra-pulmonary
TB (INDEX TB) guidelines (Sharma et al. 2017) is shown in Table 4.3.
4.6 Treatment
4.6.1 Drug-Susceptible EPTB
Treatment of EPTB is similar to treatment of pulmonary TB. Initial intensive phase

consists of rifampicin (R), isoniazid (H), pyrazinamide (Z) and ethambutol
(E) administered daily for 2 months (2RHZE). In TBM, ethambutol is replaced by
streptomycin (S) and the intensive phase would consist of 2SHRZ. In countries/
settings where the level of H resistance among new cases is high and H susceptibility
testing cannot be performed or results are not provided before the continuation phase
starts, continuation phase consists of daily RHE (Technical and Operational
Guidelines for TB Control in India 2016; Treatment of tuberculosis: guidelines.
2010; Treatment of tuberculosis. Guidelines for the treatment of drug-susceptible
tuberculosis and patient care, update 2017).
There is no universal consensus regarding the optimal duration of treatment in
patients with EPTB. Recommendations in some forms of EPTB as per the current
guidelines are as follows. The optimal duration of standard first-line treatment for
EPTB as recommended in the INDEX TB guidelines (Sharma et al. 2017) is as
follows: peripheral lymph node TB (6 months), abdominal TB (6 months) and TBM
(at least 9 months). The recently published American Thoracic Society (ATS), the
Centers for Disease Control and Prevention (CDC) and the Infectious Disease
Fig. 4.2 Diagnostic algorithm for EPTB

EPTB extrapulmonary TB, DST drug-susceptibility testing, Mtb Mycobacterium tuberculosis, S/o
suggestive of
Society of America (IDSA) guidelines (Nahid et al. 2016) state that some experts
recommend 9–12 months of treatment for bone and joint TB; 6 months of standard
treatment is considered adequate for pericardial TB and TB pleural effusion.
Table 4.3 Recommendations for use of Xpert MTB/RIF for diagnosis of EPTB as per the INDEX
TB guidelines
Pooled Pooled
Site of sensitivity specificity
EPTB (%) (%) Recommendation
Lymph Xpert MTB/RIF should be used as an additional test
node TB to conventional smear microscopy, culture and
Against 83.1 93.6 cytology in FNAC specimens (strong
culture recommendation, low-quality evidence for sensitivity
Against 81.2 99.1 estimate, high-quality evidence for specificity
CRS estimate)
TBM Xpert may be used as an adjunctive test for TBM. A
Against 80.5 97.8 negative Xpert result on a CSF specimen does not rule
culture out TBM. The decision to give anti-TB treatment
Against 62.8 98.8 should be based on clinical features and CSF profile
CRS (conditional recommendation, low-quality evidence
for sensitivity estimate, high-quality evidence for
specificity estimate)
Pleural Xpert MTB/RIF should not be used to diagnose
TB pleural TB (strong recommendation, low-quality
Against 46.4 99.1 evidence for sensitivity estimate, high-quality
culture evidence for specificity estimate)
Against 21.4 100
CRS
Data source: Sharma SK et al. (2017)
EPTB extrapulmonary tuberculosis, TB tuberculosis, CRS composite reference standard, FNAC fine
needle aspiration and cytopathological examination, TBM tuberculosis meningitis, CSF cerebrospi-
nal fluid
According to the World Health Organization (WHO) guidelines (Treatment of

tuberculosis: guidelines 2010; Treatment of tuberculosis. Guidelines for the treat-
ment of drug-susceptible tuberculosis and patient care, 2017 update 2017), 6 months
of standard anti-TB treatment is recommended for miliary TB. The WHO guidelines
(Treatment of tuberculosis: guidelines 2010) mention that some experts recommend
9–12 months of treatment for TBM and 9 months of treatment when bone and joint
TB is also present. The recent ATS, CDC and IDSA guidelines (Nahid et al. 2016)
state that 6 months of treatment is adequate in disseminated/miliary TB; these
guidelines (Nahid et al. 2016) suggest that, when associated TBM is also present,
treatment needs to be given for at least 12 months. The National Institute for Health
and Clinical Excellence (NICE) (NICE guideline (NG33) 2016) guidelines from the
United Kingdom recommend 6 months of treatment for miliary TB. In a given
patient, the duration of continuation phase may be extended as per the clinical
situation.
Patients with EPTB receiving anti-TB treatment should be carefully followed up
and monitored for adverse drug reactions, especially anti-TB drug-induced
Table 4.4 Recommendations for corticosteroid usage in some forms of EPTB as per the INDEX
TB guidelines
TB meningitis
Corticosteroids are recommended for TB meningitis in HIV-seronegative people. Duration of
steroid treatment should be for at least 4 weeks with tapering as appropriate (strong
recommendation)
Corticosteroids may be used for TB meningitis in HIV-seropositive people, where other life-
threatening opportunistic infections are absent (conditional recommendation)
TB pericarditis
Corticosteroids are recommended for HIV-seronegative and HIV-seropositive patients with TB
pericarditis with pericardial effusion (conditional recommendation)
TB pleural effusion
Corticosteroids are not routinely recommended in pleural TB (conditional recommendation)
Source: Sharma SK et al. (2017)
INDEX TB Indian Extra-pulmonary TB, EPTB extrapulmonary tuberculosis, TB tuberculosis, HIV
human immunodeficiency virus
hepatotoxicity. Patients with HIV-EPTB coinfection should be monitored for

immune reconstitution inflammatory syndrome (IRIS).
4.6.1.1 Corticosteroids
The usefulness of corticosteroids in the treatment of EPTB is not well established.
When adrenal failure is present, corticosteroids are indicated. Use of corticosteroids
can be life-saving in patients with some forms of EPTB. Recommendations for
corticosteroid use as stated in the INDEX TB guidelines (Sharma et al. 2017) are
shown in Table 4.4.
4.6.2 Drug-Resistant, Multidrug-Resistant, Extensively Drug-

Resistant EPTB and HIV-EPTB Coinfection
Drug-resistant, multidrug-resistant, extensively drug-resistant EPTB (13) and

HIV-EPTB coinfection (Nahid et al. 2016) should be treated as per standard
guidelines (Consolidated guidelines on the use of antiretroviral drugs for treating
and preventing HIV infection: recommendations for a public health approach 2016;
Antiretroviral therapy for HIV infection in adults and adolescents.
Recommendations for a public health approach 2010). A detailed description of
this is beyond the scope of this chapter.
4.6.3 Surgery
In patients with EPTB, surgery is required to procure specimens for diagnostic

testing and, in addition to anti-TB treatment, to ameliorate complications
(e.g. intestinal perforation, spinal TB, hydrocephalus). Details regarding surgical

management of EPTB are beyond the scope of this chapter.
4.7 Conclusions
A high index of clinical suspicion, use of invasive diagnostic methods for procuring
tissue, body fluids for diagnostic testing, confirmation of diagnosis by use of
molecular diagnostic tests and early institution of specific anti-TB treatment and
close clinical monitoring are key to the successful outcomes in EPTB.
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Organization, Geneva
Infections with Nontuberculous
Mycobacteria: Increased Awareness 5
and Recent Developments
Astrid Lewin and Hubert Schäfer
Abstract
Nontuberculous mycobacteria represent a multispecies and extremely heteroge-
neous group of environmental bacteria and include many species that can cause
severe disease. Still, they are given little attention as pathogenic agents, when
compared to Mycobacterium (M.) tuberculosis. Individuals at risk to become
infected by NTM usually suffer from immune defects or underlying diseases such
as cystic fibrosis or chronic obstructive pulmonary disease. Therapeutic immuno-
suppression is an additional cause for increased susceptibility, but healthy,
apparently immunocompetent individuals may also develop disease. NTM
infections can manifest as lung infections, lymphadenitis, skin, bone, and soft
tissue infections, or disseminated disease. Pulmonary disease is worldwide most
frequently caused by different species of the M. avium complex. M. abscessus
causes serious lung infections in patients afflicted by cystic fibrosis. Diagnosis of
the infection is frequently delayed because there are few specific symptoms, and
cultivation of the infective agent from clinical samples is not always successful.
Isolation of NTM from a patient’s lung, on the other hand, is no proof of
infection, but might also result from colonization of the organ or from contami-
nation with these ubiquitously occurring environmental bacteria. Therefore fur-
ther diagnostic options have to be considered before initiating antibiotic
intervention. NTM treatment is hampered by the high level of primary resistance
against antibiotics, which culminates in the multidrug resistance of M. abscessus.
New findings on infection rates, transmission routes, and nosocomial potential of
NTM are currently changing our perception of the relevance of NTM infections.
In the last two decades, an increase in NTM lung disease was reported in many
regions of the world, which can be explained by demographic trends and medical
progress prolonging life expectancy of persons vulnerable to NTM infections.
A. Lewin (*) · H. Schäfer
Robert Koch Institute, Unit 16 Mycotic and Parasitic Agents and Mycobacteria, Berlin, Germany
e-mail: lewinA@rki.de

https://doi.org/10.1007/978-981-32-9413-4_5
56 A. Lewin and H. Schäfer
Transmission of M. abscessus between patients with cystic fibrosis has

challenged the hitherto commonly accepted classification of NTM as environ-
mental non-transmissible infectious agents. And finally the incidence of severe
and fatal nosocomial infections has increased attention for NTM. Studies on the
burden of disease considering infection numbers, mortality rates, and treatment
costs have further substantiated the significance of NTM infections for public
health. In view of these new perspectives, a higher priority for NTM disease both
in basic research and when deciding on public health measures is indicated.
Keywords
Nontuberculous mycobacteria · Burden of disease · Public health impact ·
Nosocomial infections · Patient-to-patient transmission · Immunodiagnosis ·
Immunocompromised · Virulence
Abbreviations
NTM Nontuberculous mycobacteria

MAC Mycobacterium avium complex
CF Cystic fibrosis
COPD Chronic obstructive pulmonary disease
IFN Interferon
IL Interleukin
PCR Polymerase chain reaction
RFLA Restriction fragment length analysis
IS Insertion sequences
MALDI-ToF MS Matrix-assisted laser desorption ionization-time of flight mass
spectroscopy
TST Tuberculin skin test
IGRA Interferon-gamma release assay
ELISA Enzyme-linked immunosorbent assay
TB Tuberculosis
ATS American Thoracic Society
BTS British Thoracic Society
DST Drug susceptibility testing
MIC Minimal inhibitory concentration
rRNA Ribosomal RNA
GPL Glycopeptidolipids
5 Infections with Nontuberculous Mycobacteria: Increased Awareness and. . . 57
5.1 The Public Health Attention to Nontuberculous

Mycobacteria
The number of species in the genus Mycobacterium is continually expanding and

currently comprises around 200 species (Parrish 2019), the vast majority of which
belong to the group of nontuberculous mycobacteria (NTM). Although NTM have
attracted increasing attention in the last two decades, they are still considered fairly
insignificant pathogens. The dominance of M. tuberculosis within the genus Myco-
bacterium is also reflected by designating NTM as “mycobacteria other than tuber-
culosis” (MOTT) and “atypical mycobacteria”. As a consequence research activities
are focused on M. tuberculosis. Even among health professionals, NTM are rather
taken as a marginal nuisance, than an infective threat. Undoubtedly, M. tuberculosis
causing around 1.3 million deaths per year is the most important infectious bacte-
rium worldwide. However, this should not lead to negligence toward other infectious
mycobacteria. In this chapter we will point out some of the reasons for the small
attention given to NTM infections and challenge the perception that NTM are of low
significance for public health.
5.2 Environmental Habitat and Intracellular Lifestyle
Evolutionary early forms of mycobacteria probably inhabited a number of environ-

mental niches as saprophytes. Different species of the genus Mycobacterium have
adapted to various degrees of host invasion and even host dependence.
M. tuberculosis is a completely host-dependent pathogen, which has no environ-
mental niche. Obviously this species has traded in most of the skills needed during
environmental persistence and replication for capabilities that confer host adaption,
intracellular lifestyle, immune evasion and human-to-human transmission (Banuls
et al. 2015; Behr 2013). Similarly, M. avium subsp. paratuberculosis, a frequent
pathogen in ruminants, is considered an obligate parasite of animals, meaning that its
survival outside the host animal is finite (Eppleston et al. 2014). The obligate
intracellular pathogen M. leprae has undergone extensive evolutionary genome
reduction hinging these bacteria on host cell metabolism (Cole et al. 2001). These
species represent the completely host-adapted end of mycobacterial lifestyle spec-
trum. The evolutionary changes leading to this adoption can be readily observed in
their genomes (Rahman et al. 2014).
Basically all of the NTM species addressed in this chapter can both be considered
as environmental mycobacteria and opportunistic pathogens. In some cases the
distinction between mainly environmental and predominantly host dependent is
quite narrow. Strains of the bird-pathogen M. avium subsp. avium, for example,
are frequently found in surface waters, but it is not known whether all strains actively
multiply in this environment or if some pathogenic strains get there only by excretion
from birds. Similarly M. avium subsp. hominissuis is frequently found in household
Fig. 5.1 Simplified overview of the taxonomy of the genus Mycobacterium
dust (Lahiri et al. 2014), but it is very unlikely that it grows there; other frequent
NTM sources are shower heads, water pipes, and tap water (Falkinham 2016), but
again it is not certain that those niches represent the main habitat of these NTM.
Although the distinction between tuberculous and nontuberculous mycobacteria is
quite clear (Fig. 5.1), the taxonomy of the latter is still open to debate.
Several attempts have been made to subdivide NTM into different groups
according to systemic relatedness or physiological features such as growth speed
or pigmentation (Runyon 1959). Most of these subgroups however show only little
accordance of their individual members regarding pathogenicity or preference for
the site of infection and certain predispositions in the hosts. Many of the NTM
species so far identified as pathogens can cause opportunistic infections in various
tissues of immunocompromised or otherwise predisposed hosts, although there is a
certain preference for both aspects in most of the species (Table 5.1).
On the far end of the pathogenicity scale, we find species that have never been
described as pathogens and thus live purely environmental, as commensals or as
saprophytes (Primm et al. 2004). Some species that invade protozoans may be
regarded as symbionts (Yu et al. 2007), but it has not been shown what the advantage
for the protozoan partner would be.
Table 5.1 Examples for NTM species that cause disease in humans
Species Disease(s) Risk group (affected individuals) Growth
M. avium (a) Lymphadenitis (a) Young children Slow
complex (b, c and d) Lung disease (b) Immunocompromised adults
(e) Disseminated disease (c) COPD patients
(d) CF patients
(e) AIDS patients
M. abscessus (a) Lung disease (a) CF patients Rapid
complex (b) Skin and soft tissue (b) Patients exposed to contaminated
infection material after traumatic injury or
surgery
M. marinum Skin and soft tissue Recreational and professional Slow
infection handling of fish
M. kansasii Lung disease Patients suffering from silicosis Slow
(mine workers)
M. scrofulaceum Lymphadenitis Young children Slow
M. malmoense (a) Lung disease (a) Immunocompromised adults Slow
(b) Lymphadenitis (b) Young children
M. fortuitum Skin and soft tissue Patients exposed intra- or Rapid
infection postoperatively to contaminated
Wound infection instruments, water, soil, or air
M. xenopi Lung disease Immunosuppressed individuals Slow
Bone infection
M. chimaera Invasive infection Patients exposed to M. chimaera Slow
aerosols under open heart surgery
5.3 NTM Infections, Diagnosis, and Treatment
5.3.1 Predisposing Conditions and Risk Factors
Corresponding to the mainly opportunistic nature of NTM infections, most affected

patients can be grouped into three categories of predisposition or risk of infection:
• Immunodeficiency, primarily of the cellular immune response (Casanova 2001,

2015; Casanova and Abel 2002; Doffinger et al. 2000; Doffinger et al. 2002;
Reichenbach et al. 2001; Serour et al. 2007), and in particular the IFN-gamma/
IL12 axis (Jouanguy et al. 1999). Immunosuppression in transplant patients
(Knoll 2014) or treatment with anti-TNF-alpha to reduce inflammation in
rheumatoid arthritis (van Ingen et al. 2008). People suffering from acquired
immunodeficiency, such as HIV-infected and AIDS patients, were commonly
affected by NTM before the widespread practice of HAART (El-Solh et al.
1998; Liao et al. 2004).
• Patients suffering from underlying diseases that affect the lung, mainly chronic
obstructive pulmonary disease (COPD) (Char et al. 2014) or cystic fibrosis (CF),
are often diagnosed with pulmonary NTM infections (Adjemian et al. 2018).
Patients treated for lung cancer sometimes exhibit comorbidity with MAC lung
infection (Daley and Iseman 2012).
• Occupational exposure to dust and working in mines can favor NTM infection
especially with M. kansasii (Chobot et al. 1997; Policard et al. 1967). Profes-
sional handling of fish or leisure activities leading to frequent contact with fish
or contaminated water may result in skin infections with M. marinum (Gray
et al. 1990).
Persons without known immune defect or no obvious underlying disease may

acquire NTM infections too. In the USA an increasing and large group of NTM
patients are postmenopausal women above 50 years of age, with a low BMI and
above-average height and frequently with pectus excavatum, scoliosis, and mitral
valve prolapse (Chan and Iseman 2010). The concordant anatomical phenotype of
these patients however indicates a shared, but so far unrecognized, hereditary
predisposition.
Extrapulmonary infections in subjects with no known predisposing factors
include lymphadenitis in small children, but closer scrutiny and additional studies
in infection epidemiology might reveal hereditary or exposure-related risks (Garcia-
Marcos et al. 2017; Serour et al. 2007).
5.3.2 Diagnosis of NTM Infections
The diagnosis of NTM infections is largely dependent on the organ system involved.
The “gold standard” is represented by cultivation and identification of the infectious
agent in samples taken from the site of infection.
Molecular tests are available now to identify NTM to species and subspecies
levels. They involve hybridization techniques, PCR (where appropriate combined
with RFLA), and gene-sequencing techniques as well as whole genome sequencing
(reviewed in Koh 2017; O’Connor et al. 2015; Ryu et al. 2016; Slany and Pavlik
2012). Additionally, MALDI-TOF offers a promising approach for species and even
subspecies determination (O’Connor et al. 2015; Alcaide et al. 2018), although this
method is not yet established for use in routine diagnosis of NTM and still needs
further development.
The hybridization techniques such as the line probe assays detect sequence
polymorphisms in different parts of the rRNA operons. A variety of genus-, spe-
cies-, and subspecies-specific PCRs have been developed, and some of these make
use of the presence of different insertion sequences (IS elements) in different species/
subspecies. PCR can be complemented by RFLA or sequencing of the product.
Sequencing of the 16S rRNA gene is the reference method for identification of NTM
to species or complex level, has a broad species coverage, and also identifies rarely
encountered species. If further differentiation is needed, multigene sequencing (e.g.,
of 16S-23S rRNA internal spacer ITS, rpoB, hsp65) is recommended. Whole

genome sequencing (WGS) followed by comparison with mycobacterial sequences
present in public databases is the most accurate method and in addition to species
identification provides valuable information such as presence of antibiotic resistance
genes, virulence genes, or phylogenetic relations. An overview on molecular identi-
fication techniques is presented in Table 5.2.
In contrast to M. tuberculosis infections however, where a positive culture is
readily accepted as proof of infection and reason for treatment, the situation is
Table 5.2 Techniques for molecular identification of NTM (reviewed in 32–35)

Commercial kits/
analyzed genes/
Technique components Main advantages Main disadvantages
Hybridization 16 rRNA gene Speed; ease Cross-reactivity; for a
(AccuProbe, limited number of NTM
Hologic); 16S-23S only
ITS (INNO-LiPA,
Innogenetics);
23S rRNA gene
(GenoType
Mycobacterium
CM/AS, Hain
Lifescience);
16S-23S ITS (Speed-
Oligo Mycobacteria,
Vircell);
PCR Different genus-, Specificity; Different primers for
species-, and sensitivity; ease different species/
subspecies-specific subspecies required
DNA regions (e.g.,
IS elements)
PCR followed by 16S rRNA gene; 16S rRNA gene Lengthy
sequencing of the 16S-23S ITS; rpoB sequencing is
PCR product gene; hsp65 gene; reference standard;
others (dnaJ, secA1, good discrimination
recA, sodA) level (depending on
gene selection to
subspecies level)
Whole genome Entire DNA Most accurate and Costly; lengthy;
sequencing discriminatory bioinformatics expertise
method; provides required
additional
information (e.g., on
antibiotic resistance
or virulence)
MALDI-TOF- Whole cell Speed; nonlaborious; Databases not yet
MS preparations cost-effective complete and fully
(if instrument available; lack of
available) discrimination for certain
species; lack of
standardization and
interlaboratory
reproducibility
complicated by the environmental habitat and ubiquitous occurrence of many NTM

species. Especially the microscopic and culture confirmation of NTM from the lung
is not necessarily indicative of an ongoing infection, but might result from mere
colonization of certain areas of the organ. Therefore different or additional diagnos-
tic criteria have to be considered to reach a substantiated decision. These additional
measures largely depend on the site of infection.
5.3.2.1 Pulmonary Infections

As outlined earlier, patient groups mostly affected by NTM infection of the lung
suffer either from CF, COPD, bronchiectasis, immunosuppression, immunodefi-
ciency, or reduced pulmonary function for various reasons. Although the elevated
risk for NTM infection in these patients is widely known, diagnosis is frequently
delayed, because the presentation of symptoms is not disease-specific, and the
isolation of NTM from clinical samples, although a prime demand for the diagnosis,
does not distinguish between infection, colonization, and mere contamination of the
sample (Cowman and Loebinger 2018; Catanzaro 2002). The situation is further
complicated by the different mycobacterial species that are able to infect the lung.
The first challenge is the differentiation between tuberculosis and NTM disease or
even mixed infections (Ellis 2004; Grubek-Jaworska et al. 2009; Kendall et al. 2011;
Kim et al. 2018; Kwon and Koh 2014; Pang et al. 2017; Thanachartwet et al. 2014;
Yuan et al. 2014). If M. tuberculosis infection can be excluded, it is still critical to
correctly identify the pathogen at least to the species level (Adelman and Addrizzo-
Harris 2018). Certain patient groups are more susceptible to specific species, such as
M. abscessus frequently infecting CF patients (Park and Olivier 2015), but there is
no one-to-one relation between infective agent and underlying disease, and predom-
inant pathogens differ between countries (Adelman and Addrizzo-Harris 2018).
Furthermore, even experts rarely agree on the best scheme of diagnosis and clinical
management of infection (Marras et al. 2015), and guidelines are not always
followed by physicians (El-Zeenni et al. 2018). Proof of infection does not automat-
ically indicate a need for treatment (Griffith et al. 2002); delayed treatment on the
other hand might result in severe complications and disease progression. Chest
imaging alone is not always sufficient because radiographic manifestations cannot
reliably be attributed to NTM infection and in some cases it is even hard to
distinguish this illness from lung cancer (Kobashi et al. 2004; Mohamad et al.
2012; Noguchi et al. 2016; Umehara et al. 2015). Therefore the decision to treat is
usually based on the clinical presentation, radiological features, and microbiological
findings (Aksamit et al. 2014; Kwon and Koh 2016; Ryu et al. 2016). Genetic testing
and PCR-based recognition of the pathogen is increasingly performed in specialized
laboratories (Chae et al. 2017; Hsiao et al. 2010; Jung et al. 2016; Kim et al. 2014;
Kim et al. 2018; Wang et al. 2015; Wang et al. 2017). More recently an assay has
been developed and marketed in Japan, which allows the serodiagnosis of M. avium
lung infections with sufficient sensitivity (Shu et al. 2013; Kobayashi 2014) and the
potential to distinguish between colonization and infection (Kitada et al. 2002).
Subjects without predisposing conditions are even more likely to stay undiag-
nosed for prolonged periods of time if they come up with NTM infections of the
lung. Due to the unspecific symptoms these patients present with, they frequently
have a long history of misdiagnosis and non-satisfactory treatment results, and only
experienced pulmonologists or specialists for NTM lung infections come to a fast
and reliable diagnosis. For such cases serological assays or PCR-based methods
covering a broader spectrum of potential pathogens affecting the lung should proof
very valuable.
5.3.2.2 Infections of Lymph Nodes of the Head and Neck

Cervicofacial lymphadenitis due to NTM infection is a rare but not uncommon
condition in small children. Similar to other NTM infections, the symptoms are little
specific, and differential diagnostics to exclude several infective and noninfective
etiologies have to be considered. It has been observed that needle biopsy led only in
about half of the cases to a culture-confirmed diagnosis (Willemse et al. 2018) and
this procedure was therefore not considered to sufficiently prove or rule out NTM
infection. Marks et al. (Marks et al. 1977) and Magdorf et al. (Magdorf et al. 2008)
suggested skin tests with tuberculin and sensitin, to diagnose a mycobacterial
infection and to distinguish between M. tuberculosis and NTM infection at the
same time. A recent systemic review of diagnostic studies (Willemse et al. 2018)
concluded that skin tests with sensitin-purified protein derivative (s-PPD) are the
best diagnostic option so far available, but that they should be confirmed by PCR or
culture if possible. An interferon-gamma release assay (IGRA) might provide a good
preoperative alternative, since it is noninvasive, but has not been studied in sufficient
numbers to allow reliable recommendations.
5.3.2.3 Skin, Soft Tissue, and Wound Infections

In skin and soft tissue infections, the infected area is usually readily accessible and
samples can be taken for analysis. Molecular methods such as PCR, gene sequenc-
ing, and mass spectrometry allow the rapid and detailed analysis of the infecting
agent, sometimes even without culture amplification of the bacteria (Misch et al.
2018). The main culprit leading to misdiagnosis is that NTM infections of the skin
are relatively rare and may be misjudged as a manifestation or symptom of a
predisposing condition (open wound, surgical treatment, autoimmune disease
requiring immunosuppression), instead of a consequent infection (Touma et al.
2013). Skin infections with M. marinum in association with fish tanks or profes-
sional handling of fish or marine shells have been observed at least since the 1970s
(Barrow and Hewitt 1971), and the disease is frequent enough to bear its own
designation as “fish tank infection” or “fish tank granuloma.” Most of these
infections involve the fingers and hand (Aubry et al. 2002), and therefore the
experienced dermatologist will suspect the cause of disease by the localization and
mode of infection. The microscopic or culture confirmation however is difficult,
because M. marinum requires temperatures below 30 C and several weeks to form
colonies when cultivated from patient samples (Aubry et al. 2017).
Cutaneous infections with M. abscessus massiliense have also been reported in
increasing frequency after tattooing of immunocompetent individuals. The source is
probably contaminated ink or dilution of ink with non-sterile water. Surgery-related
infections (see below) frequently involve skin and soft tissue and thus might be
superficial or at least in the early stage restricted to the area affected by surgery.
Diagnostic options therefore are isolation and culture of the pathogen.
5.3.2.4 Ocular Infections

In ocular NTM infections, early diagnosis and recognition of the infections agent is
crucial for the outcome of the treatment (Kheir et al. 2015). Besides M. tuberculosis
(Albert and Raven 2016), a number of additional mycobacterial species have been
reported to infect the eye (Chu et al. 2015). As in many other NTM infections,
diagnosis is often delayed, and hence treatment and management of the disease are in
many instances far from optimal (Patel et al. 2013).
5.3.2.5 Immunodiagnosis
Both cellular and humoral immune responses can be used as starting points for the
diagnosis of mycobacterial infections.
Cellular immunodiagnosis, applied in vivo as the tuberculin skin test (TST), is a
well-established routine procedure to reveal contact with M. tuberculosis, including
recent and latent infections which have not led to symptoms of disease. The TST
however does not discriminate between active disease and latent infection or even
cured disease. Earlier vaccination with Bacillus Calmette-Guerin (BCG) might also
lead to a positive result. Therefore confirmation of infection is required by either
mycobacteria-positive sputum smears or radiographic evidence for lung disease or
extrapulmonary manifestations. IGRAs can be considered as an advanced in vitro
version of the TST and carry several improvements: firstly the antigens used are
specific for M. tuberculosis and are not cross-reactive with BCG and most NTM
species; secondly, the test does not include injection of mycobacterial antigen; and
thirdly results are obtained in the laboratory, circumventing the need for a second
visit with the physician to assess the result (Thillai et al. 2014). In parallel to the TST,
the IGRA cannot distinguish between latent or cured infection and active disease, but
at least in low-incidence countries, it is recommended for the diagnosis of latent TB
(WHO 2018) and screening of contacts.
Serological assays for TB infection, although commercially available in various
forms, are not recommended by the WHO (WHO 2010), due to highly variable
values for sensitivity and specificity. These recommendations have probably
affected the development and use of serological assays for detection of NTM
infections.
As mentioned earlier, two different approaches for the immunodiagnosis of NTM
infections are used to a certain extent. The cellular approach was to transfer the TST
principle of tuberculosis to different mycobacterial species and mycobacterial
infections in humans and animals (Magnusson 1961) and to address sensitivity
and specificity in different species (Magnusson et al. 1961; Magnusson 1961).
PPD preparations of NTM were commonly designated as “sensitins” (s-PPD)
independently of the actual source. The use of M. avium sensitin turned out to be
useful for detection of M. avium infections in humans, despite intense cross-

reactions with tuberculin (Fordham von Reyn et al. 1995). Generally, the ubiquitous
pervasiveness of environmental mycobacteria may lead to sensitization of healthy
and diseased individuals, so independently from cross-reactions with
M. tuberculosis, a positive test result for s-PPD might not reflect current or preceding
infection. Although the debate on the use and the value of this diagnostic tool is still
going on, a recent review of the published literature attested sufficient specificity and
sensitivity to use the s-PPD skin test as a first assay, which, if positive, has to be
confirmed by culture or PCR methods (Willemse et al. 2018). Either of the latter
measures is indispensable, because it will also provide details of the pathogen which
are important to decide on treatment options. More recently the s-PPD skin test has
also been used in its in vitro equivalent IGRA (Steindor et al. 2015) for the detection
of NTM infections, but published data are not sufficient to come to a conclusion if
this is actually an improvement.
In the absence of an all-purpose gold standard for the diagnosis of NTM
infections, serodiagnosis tailored to detect immune reactions against specific
pathogens might provide a valuable tool to assist in the differential diagnosis of
infections with nonspecific symptoms. Commercial kits for the diagnosis of bovine
M. avium subsp. paratuberculosis are available, but upon closer evaluation none of
the assays turned out to be fully satisfactory (Kohler et al. 2008).
Starting in 2002, Kobayashi and co-workers suggested and developed the use of
glycopeptidolipids (GPL) from M. avium as a diagnostic antigen for M. avium
infections in humans (Kitada et al. 2002). It had been well known that GPL represent
one of the immunodominant antigens in several mycobacterial species (Ikawa et al.
1989; Schaefer 1967), but the expression of different variations, due to diverse
glycosylation patterns, even between different strains of the same subspecies, is a
challenge for the diagnosis of M. avium infections in particular. The advantage of
using GPL as diagnostic antigens resides in the absence of these cell wall
components in tuberculous mycobacteria, thus ruling out cross-reactions with TB
infections. After several refinements, the test was brought to the market (Kobayashi
2014) and is further evaluated in different settings and patient populations (Kitada
et al. 2010, 2015, 2017; Shu et al. 2013). The problem of the highly variable GPL
glycosylation was solved by using the GPL core antigen as a diagnostic target in
M. avium infection, but it will have to be shown whether the same concept can be
applied to the specific diagnosis of infections with other NTM.
In conclusion, both IGRA and ELISA might provide valuable tools for noninva-
sive diagnosis of suspected mycobacterial infections, and a consequent development
and use of such assays should help to shorten the lack time between appearance of
symptoms and treatment start in various settings.
5.3.3 Treatment
Compared to M. tuberculosis disease, there is no room for empirical treatment when

NTM infection is suspected. Before initiation of treatment, clinical, radiological, and
microbiological features must be taken into consideration, and ATS and BTS
guidelines should be followed as closely as possible. The management options of
mycobacterial infections are basically limited to antibiotic treatment and surgical
removal of infected tissue. Surgery is quite successful for the cure of lymphadenitis
at the head and neck in children (Lindeboom et al. 2007), although not without risk
of permanent damage to facial nerves and of ill-favored scars of affected areas
(Gonzalez et al. 2016). Excision of infected tissue is also an option for refractory
lung disease (Griffith and Aksamit 2012; Sakane et al. 2018), if other treatment
options have failed and localization of the infection permits surgical intervention.
The treatment strategies that need to be applied to eliminate NTM or reduce NTM
load depend on the interplay of NTM species, patient characteristics, and disease
manifestations. Mycobacteria can display considerable resistance toward antibiotics
(Nasiri et al. 2017), and especially the multidrug-resistant M. abscessus is considered
an “antibiotic nightmare” (Nessar et al. 2012). Antibiotic therapy is complicated by
the combination of a number of independent obstacles that frequently occur in NTM
infections: (i) primary antibiotic resistance of environmental mycobacteria;
(ii) additional acquired resistance under treatment; (iii) serious, sometimes fatal
side effects of the remaining effective antibiotics; (iv) long (months to years)
treatment time needed for conversion; (v) coinfections with other bacteria or fungal
pathogens; and (vi) underlying disease of the patients, including immunodeficiency
or need for immunosuppression.
The design of appropriate treatment strategies is also hampered by difficulties in
drug susceptibility testing (DST). Broth microdilution assays such as the Sensititre
System (Trek Diagnostics) are suitable for determination of minimal inhibitory
concentration (MIC). The protocol from the Clinical and Laboratory Standards
Institute (Clinical and Laboratory Standards Institute M24-A2, 2011) (Clinical and
Laboratory Standards Institute M62, 2018) proposes interpretations of MIC values
for certain antibiotics and currently is the gold standard for DST of nontuberculous
mycobacteria. A major problem in treatment of NTM infections is the lack of
correlation between results of DST and clinical outcome (van Ingen and Kuijper
2014). Exceptions to this are amikacin and macrolides, whose MIC values correlate
well with the clinical outcome in treatment of lung infections by MAC (Van Ingen
et al. 2012; Brown-Elliott et al. 2013; Bastian et al. 2011; Prammananan et al. 1998).
Macrolides and amikacin furthermore stand out because their efficacy against certain
NTM can be predicted by molecular analysis of their target genes. Thus macrolide
susceptibility of MAC and M. abscessus can be assessed by sequencing of the 23S
rRNA gene (Griffith et al. 2006; Bastian et al. 2011), while sequencing of the 16S
rRNA gene provides information on amikacin susceptibility (Brown-Elliott et al.
2013; Prammananan et al. 1998). In addition M. abscessus may have complete or
truncated versions of the erm(41) gene causing inducible macrolide resistance
(Zheng et al. 2019). DST of M. abscessus strains by broth microdilution assays
should include additional plate reading after up to 14 days in order to detect
inducible macrolide resistance (Clinical and Laboratory Standards Institute
M24-A2, 2011) (Clinical and Laboratory Standards Institute M62, 2018). Analysis
of the molecular structure of the erm(41) gene from M. abscessus can also be used
for subspecies identification, because the subspecies M. abscessus massiliense is
distinguished from the other subspecies by a truncated nonfunctional erm(41) gene
(Adekambi et al. 2017). Next to gene or genome sequencing, the commercial line
probe assay GenoType NTM-DR (Hain Lifescience) provides a molecular assay for
detection of amikacin and macrolide resistance in MAC and M. abscessus.
Although guidelines exist for the management of NTM infections in different
countries and patient groups (Haworth et al. 2017; Haworth and Floto 2017;
McGrath et al. 2008; Floto et al. 2016; Griffith et al. 2007), the factors listed
above may limit adherence to general recommendations. Experts however criticize
the lax interpretation of relevant guidelines and suppose that a more complete
implementation will result in better treatment outcome for most of the diseased
(El-Zeenni et al. 2018).
5.4 Obstacles in Evaluating the Public Health Impact

of Nontuberculous Mycobacteria
Why is it problematic to assess the role of NTM infections for public health? The
main reason is the diversity of NTM species, of hereditary predispositions within
patient groups concerned, and of disease manifestations. This diversity prevents us
from viewing and evaluating NTM as a whole.
First of all, their growth properties differ substantially leading to division into
slow and rapid growers according to generation time. Species generating visible
colonies on agar within 7 days belong to the rapid growers; the remaining ones are
slow growers. There is a correlation between growth rate and pathogenicity: slow-
growing mycobacteria are frequently more pathogenic (Lewin and Sharbati-Tehrani
2005). An exception to this rule is M. abscessus, a rapid-growing Mycobacterium of
high clinical significance particularly in CF patients (Adjemian et al. 2018). NTM
diversity does not end at species level. Even within the same species, different
colony morphologies can occur that impact host-pathogen interaction (Gutierrez
et al. 2018) and disease severity. NTM inhabit different environments such as
water, soil, dust, and amoeba (Samba-Louaka et al. 2018) and can form biofilms
on tissues, transplants, and medical devices (Ghosh et al. 2017). Generally, they are
opportunistic pathogens but some species can also infect immunocompetent persons
(Piersimoni and Scarparo 2009). As described above NTM cause a variety of
diseases.
Owing to this complexity, most studies focus on selected conditions of NTM
disease limiting their validity for the assessment of the public health impact of NTM.
5.5 New Developments Demanding a Re-evaluation of NTM
5.5.1 NTM Infection Rates
There is much debate on the question if infections by NTM are indeed increasing in
incidence or if reports on increasing frequencies of NTM isolation and disease rather
reflect a rise in awareness and improved diagnostic techniques. It is difficult to
answer this question, because NTM are non-reportable diseases in most countries of
the world, hampering the availability of complete infection numbers (Henkle et al.
2015). As a consequence numerous regional studies are performed that follow
region-specific aims and differ therefore with respect to variables such as (i) the
quantified epidemiologic measure (incidence, prevalence, or isolation numbers),
(ii) the investigated diseases, (iii) the included patient groups, (iv) the geographical
regions, and (v) the time periods included. These limitations impede comparability
and definition of generally accepted trends. The majority of these studies conclude
that NTM infections are increasing, and we refer to recent reviews on this topic
(Johnson and Odell 2014; Prevots et al. 2010; Strollo et al. 2015). For example, a
study comprising the US Medicare Beneficiaries came to the conclusion that the
annual prevalence of NTM pulmonary infections increased from 1997 to 2007 from
20 to 47 cases/100,000 population or 8.2% per year (Adjemian et al. 2012).
Especially studies on NTM lung infections report increasing infection rates, while
extrapulmonary NTM infections do not show this effect. Different trends for
pulmonary vs. extrapulmonary infections were also supported by data from Oregon,
USA. While the incidence of extrapulmonary diseases in Oregon remained stable at
around 1.5/100,000 population from 2007 until 2012 (Henkle et al. 2017), lung
infections increased from 4.8/100,000 to 5.6/100,000 in the same period (Henkle
et al. 2015). The only geographic region with a notification system for all NTM
infections is Queensland, Australia, where from 2012 to 2015 a yearly increase in
notification rates of approximately 17% was observed resulting in a notification of
25.9 cases per 100,000 population in 2015 (Thomson et al. 2017).
Altogether, there is sufficient body of evidence to support the assumption that,
despite regional differences, there is an increasing trend in pulmonary NTM
infections. These increasing NTM infection rates can be explained mainly by
demographic trends and medical progress prolonging life expectancy of persons
vulnerable to NTM infections.
5.5.2 Correlation Between Virulence and Genome Properties
Clinically relevant NTM are opportunistic pathogens, while host susceptibility

certainly is the predominating condition determining if colonization with NTM
will end in disease or not (Henkle and Winthrop 2015). The contribution of genomic
features of different NTM species to cause illness (i.e., virulence factors) is still
under study. Increasing use of whole genome sequencing in combination with
association of clinical data should provide answers to this question.
Comparative genome analysis of M. avium strains provides first indications for

the existence of bacterial genomic determinants for disease progression. Most
studies performing cluster analysis with NTM genomes were conducted with
genomes from M. avium. M. avium subsp. hominissuis is the clinically most impor-
tant and also the genetically most variable of the four M. avium subspecies (Rindi
and Garzelli 2014). The main determinants of genomic variation in M. avium subsp.
hominissuis are small nucleotide polymorphisms (SNP), along with large sequence
polymorphisms (LSP), minisatellite sequence polymorphisms, and the distribution
of insertion elements (Rindi and Garzelli 2014). M. avium subsp. hominissuis has an
open pan genome (the entire gene set of all strains of a species) supporting the
genomic diversity of this subspecies (Uchiya et al. 2017). When comparing the
genomes of a collection of 41 M. avium subsp. hominissuis containing 20 clinical
and 21 environmental isolates from Germany, Sanchini and colleagues identified a
new hypervariable genomic island that could be further divided into 8 different
genome island types comprising 253 different genes in total. However, their analysis
did not show any correlation between presence or absence of certain types of this
hypervariable genomic island and habitat of the isolates (Sanchini et al. 2016). The
same strain collection was used to investigate the capacity of the strains for
conjugative plasmid uptake. Interestingly, it was found that environmental isolates
are more proficient in conjugative DNA uptake than clinical isolates and also
contained a higher proportion of plasmid-derived genes (Shoulah et al. 2018). The
capacity to act as conjugative recipient was correlated with absence of or mutations
in a genomic DNA region containing the gene radC and the genes for components of
a type I restriction/modification system. This region was differently organized in
environmental compared to clinical isolates (Shoulah et al. 2018) pointing to its
potential as marker predicting pathogenic potential. The results obtained with the
strain collection of M. avium subsp. hominissuis from Germany differed from those
reached by the use of a strain collection from Japan (Uchiya et al. 2017). Phyloge-
netic analysis of genome sequences from 46 clinical M. avium subsp. hominissuis
isolates from Japan, 17 of which were classified as causing progressive disease and
29 as causing stable disease, positioned the isolates belonging to the progressive
disease group in one cluster. DNA regions classified as “hot spot regions for
mutation and/or recombination” could be identified in the core genomes. The
noncore sequences of the progressive-type strains contained cluster-specific genes
including known virulence-related genes. The plasmid pTH135 belonged to the
noncore sequences of the progressive strain cluster, suggesting a contribution of
this plasmid to virulence (Uchiya et al. 2017). This result is opposed to the result
from Shoulah et al. (Shoulah et al. 2018), who found less genes from plasmids
related to pTH135 in clinical isolates from Germany compared to environmental
ones. Geographic strain variation may explain diverging results obtained with the
German and Japanese isolates.
Progress in differentiating isolates with varying virulence potential has also been
achieved for M. abscessus. A population-level study involving whole genome
sequencing revealed existence of global transmissible M. abscessus clusters
displaying increased antibiotic resistance, rates of chronic infection, phagocytic
uptake, and intracellular survival in macrophages as well as greater bacterial burden

upon infection of severe combined immunodeficient (SCID) mice (Bryant et al.
2016). Identification of markers for these particularly virulent clusters in the future
may assist in the decision on the need for treatment in order to prevent chronic
M. abscessus infections.
In summary, these studies suggest that bacterial virulence factors noticeably add
to the progression of colonization by NTM to disease. Once these factors are
determined, their use in diagnostics should support prognosis of disease progression
and decision on the need for therapy.
5.5.3 Nosocomial Infection and Patient-to-Patient Transmission

of NTM
NTM are environmental bacteria and can be isolated from water, soil, dust, and
biofilms (Halstrom et al. 2015). They also survive in amoeba (Sharbati-Tehrani et al.
2005; Ovrutsky et al. 2013) and infect or colonize animals (Biet and Boschiroli
2014; Keller et al. 2018). Although more rarely, food was also shown to be
contaminated by NTM (Cerna-Cortes et al. 2009). Finally infected humans represent
a niche for NTM survival and growth.
The aforementioned habitats of NTM represent possible reservoirs for human
infections. Proof of transmission requires molecular typing of the isolates from the
patients and the potential infection source. Typing methods involve, for example,
variable number tandem repeat typing (VNTR) (Torres-Coy et al. 2016), random
amplification of polymorphic DNA (RAPD-PCR) (Sax et al. 2015), pulse field gel
electrophoresis (PFGE) (De Groote et al. 2006), or whole genome sequencing
combined with single nucleotide polymorphism (SNP) analysis, which has the best
discriminatory power (Trovato et al. 2017) and is indeed the only method that can
prove transmission routes beyond doubt.
As NTM are most abundant in environmental habitats, the environment is probably
the most important source of infection. NTM are present in natural water reservoirs as
well as in drinking water (Makovcova et al. 2014). Especially tap water has been
frequently associated with NTM infection (Falkinham 2016). Resistance toward water
disinfection procedures supports the survival and spread of NTM in drinking and
municipal water systems. M. avium bacteria, for example, display high resistance
against chlorine, monochloramine, chlorine dioxide, and ozone, and their calculated
disinfection values (CT99.9%) for chlorine were shown to be 580–2300 times greater
than those of Escherichia coli (Taylor et al. 2000). The hydrophobicity of the
mycobacterial cell wall supports their adherence to surfaces including household
plumbing materials or medical devices followed by the formation of biofilms.
Biofilm-grown M. avium can reach densities as high as 107 CFU/cm2 (Mullis and
Falkinham 2013). They withstand flushing and are further protected against disinfec-
tant activity. Oriani and colleagues tested the tolerance of M. gordonae and
M. chubuense to the disinfectant sodium hypochlorite. Both NTM species when
grown in biofilms require treatment at a concentration of 10 ppm for 60 min to achieve
significant reduction of CFU, while a similar reduction was reached with a concentra-
tion of 2 ppm (M. gordonae) or 5 ppm (M. chubuense) during the same time period
when planktonic cells were treated (Oriani et al. 2018). Resistance to disinfectants and
biofilm formation are key elements for the success of waterborne NTM as infectious
agents. This is especially true for those NTM belonging to the M. avium complex
(MAC), which was repeatedly associated with waterborne infections involving use of
tap water, for example, in hot tubs (Kahana et al. 1997) or during showering
(Falkinham et al. 2008) [reviewed in, e.g., Halstrom et al. 2015].
Ecological studies involving isolation of NTM from environmental samples
underline the importance of habitats other than water as infection sources. For
example, Lahiri et al. (Lahiri et al. 2014) analyzed 130 environmental samples for
the presence of cultivable M. avium subsp. hominissuis and found these in 20% of
soil samples and 33% of dust samples but in none of the water samples. Dust and
aerosolized soil should therefore be taken into consideration when searching for
NTM infection sources and designing prevention measures. De Groote and
colleagues succeeded in recovering M. avium from a patient’s potting soil and
linking it to the patient’s isolate by pulse field gel electrophoresis (PFGE)
(De Groote et al. 2006).
The relevance of zoonotic NTM infections is still not clear. Zoonotic
transmissions are well known to occur by handling of fish or aquarium water. The
most important NTM in this respect is M. marinum, which can penetrate the skin via
minor traumata (Vincenzi et al. 1992; Keller et al. 2018; Gray et al. 1990). The nine-
banded armadillo is known to represent a reservoir for M. leprae, and there is
evidence of transmission to humans in Florida and Brazil (da Silva et al. 2018;
Domozych et al. 2016). Possums and mosquitoes are suggested as zoonotic
reservoirs of M. ulcerans (causing Buruli ulcer) in southeast Australia (Huang and
Johnson 2014). The potential role of M. avium subsp. paratuberculosis, which
causes Johne’s disease in ruminants, as a causative agent of Crohn’s disease in
humans is still under debate (Waddell et al. 2008; Garvey 2018; Atreya et al. 2014).
The presence of NTM in a wide number of animal species (Biet and Boschiroli 2014)
suggests more frequent zoonotic transmission; the final proof by molecular typing
has, however, only rarely been attempted. More frequent use of next-generation
sequencing will shed new light on this still unresolved question.
The presence of NTM in livestock and animal-derived products suggests the
occurrence of foodborne infections. NTM or DNA from NTM were found in milk
products (Leite et al. 2003) and in powdered infant formula, ground beef, chicken
sausage, and mortadella cold cut (Sevilla et al. 2017). Also products from fruits and
vegetables/salads were shown to contain NTM, such as orange juice (Cerna-Cortes
et al. 2016), salad (Cerna-Cortes et al. 2015), or chili sauce (Cerna-Cortes et al.
2009). Among the NTM found in animal-derived food products, M. avium seems to
be prevailing (Sevilla et al. 2017; Klanicova-Zalewska and Slana 2014). Molecular
typing by VNTR and IS1245 RFLP had revealed similarities between M. avium
subsp. hominissuis isolates of human and porcine origin (Leao et al. 2014;
Tirkkonen et al. 2007) suggesting either transmission between humans and pigs or
infection by the same environmental source.
In medical settings, NTM species which are capable to withstand water disinfec-
tion procedures and which can form biofilms on the one side and persons vulnerable
to NTM on the other side confront each other. In a study comparing occurrence of
NTM in hospitals and home water systems, higher isolation rates were found in
hospitals (Peters et al. 1995). Specific NTM strains seem to have the capacity for
long-term persistence in hospital water supplies as shown by Crago et al. (Crago
et al. 2014), who monitored NTM in a hospital water distribution system in Alberta
and found the same M. avium strain in 34/183 sampling sites over 2.5 years. An
overview on waterborne nosocomial infections is presented by Li et al. (Li et al.
2017). The authors of this review calculated the median attack rate to be 12.1% with
a low mortality rate (Li et al. 2017). Other sources of nosocomial NTM infections
can be devices (e.g., heater-cooler units, extracorporeal membrane oxygenation
devices, endoscopes), surgical sources (e.g., in cosmetic and eye surgery), and
aqueous solutions (e.g., use of contaminated ultrasound gel), reviewed in Sood
and Parrish (2017). The increasing popularity of cosmetic surgical procedures,
including cutaneous surgery, mesotherapy, liposuction, and laser resurfacing, leads
to a rising incidence of NTM infections in otherwise healthy people (Green et al.
2017; Avanzi et al. 2018; Berliner et al. 2015; Chang et al. 2017; Torres-Coy et al.
2016). The manifestation is mostly in skin and soft tissues and the options of
diagnosis have been described above. Transplant recipients are at high risk of
NTM infections because they receive immunosuppressive treatment after surgery
(Daley 2009; Huang et al. 2011; Jankovic Makek et al. 2016; Pandian et al. 2008;
Patel et al. 1994; Piersimoni 2012; Rao and Silveira 2018; Shah et al. 2016).
Recipients of allogeneic stem cell transplants are even more susceptible to NTM
infections, which in most cases affect the lung (Beswick et al. 2018; Doucette and
Fishman 2004; Knoll 2014). The frequently delayed identification of the infectious
agents contributes to enhanced morbidity and mortality in this highly vulnerable
patient population.
Since 2013 a global outbreak (Europe, Australia, USA), involving more than
100 patients, caused by M. chimaera present in heater-cooler devices used during
heart surgery has attracted much attention (Sax et al. 2015; van Ingen et al. 2017).
Persistence of M. chimera in the heater-cooler devices due to their tolerance to the
disinfection protocols applied (Walker et al. 2017) in connection with biofilm
formation and aerosolization into the operation area caused this outbreak. The
patients suffered from prosthetic valve endocarditis, sternal wound infection, aortic
graft infection, and disseminated disease. An evaluation of the disease progression of
the first 30 UK cases revealed a mortality rate of 60% (Scriven et al. 2018). The
likely source of this outbreak is a point-source contamination of the heater-cooler
devices at the factory (van Ingen et al. 2017). This outbreak very clearly emphasizes
the importance of NTM as nosocomial pathogens.
Until recently NTM were assumed to be non-transmissible between humans. In
2012 however, a report on probable transmission of M. abscessus subsp. massiliense
between patients in a CF center was published (Aitken et al. 2012). The
methodologies applied to compare the strains were PFGE and repetitive unit-
sequence-based polymerase chain reaction, which may not have the discriminatory
power to prove inter-patient transmission. PFGE was also used in a study that
compared M. abscessus isolates from an outbreak among CF patients in Hawaii in
2012 and suggested patient-to-patient transmission (Johnston et al. 2016). In 2013,
Bryant and colleagues could prove transmissions between patients of M. abscessus
subsp. massiliense in a CF center in the UK by whole genome sequencing (Bryant
et al. 2013). Further reports on possible transmissions of M. abscessus between
patients with CF followed (Kapnadak et al. 2016). The dimension of the occurrence
of patient-to-patient transmission of M. abscessus was finally demonstrated by a
study comparing whole genome sequences of 1080 clinical isolates from 517 CF
patients from Europe, the USA, and Australia. Phylogenetic analysis illustrated the
presence of three human-transmissible global M. abscessus clusters that displayed
increased adaptation to the human host (Bryant et al. 2016). This study has changed
our view on NTM from a purely environmental to a human-transmissible group of
bacteria. However, it is intriguing that all human-to-human transmissions so far
described only concern CF patients and M. abscessus bacteria. Further epidemio-
logic studies including whole genome sequencing and cluster analysis are needed to
work out if transmission between humans is a peculiarity of M. abscessus infections
in CF patients or if it indicates a more general transmission path of nontuberculous
mycobacteria.
5.6 Burden of Disease
As aforesaid, NTM infections are not reportable in most countries in the world,
making it problematic to achieve sufficient data to fully describe the burden of
disease.
The prevalence of NTM pulmonary infections is increasing in many areas of the
world, and in certain geographic areas, it even exceeds the prevalence of pulmonary
tuberculosis. When comparing prevalences of pulmonary NTM and tuberculosis in
the 3-year period from 2004 to 2006 in four integrated healthcare delivery systems in
the USA, the prevalence of pulmonary NTM disease was 2–2.9-fold higher than the
tuberculosis prevalence (Prevots et al. 2010). This study also demonstrated that this
increase mainly resulted from infections in the age group 80 years and older and
within this age group women contributed more than men. It can be expected that the
current demographic trend toward an aging population will further increase the
prevalence of pulmonary NTM infections. The second driving force behind increas-
ing NTM prevalence is the medical progress enhancing the life expectancy of
vulnerable patient groups such as those with CF or COPD. The life expectancy for
persons with CF, for example, is steadily increasing, and a UK study has shown that
predictions of a mean survival of >50 years of age for infants born in the UK since
the year 2000 are realistic (Dodge et al. 2007). As the percentage of CF patients with
NTM increases with age (Adjemian et al. 2018), the longer life expectancy of CF
patients will lead to increasing NTM prevalence rates. The prevalence of COPD,
another risk factor for acquiring NTM pulmonary disease, is also increasing. In a
study comparing and predicting the ranking of certain diseases with respect to their
contribution to disease burden [measured in disability-adjusted life years (DALYs)]

in 1990 and 2020, COPD was predicted to move from place 12 in 1990 to place 5 in
2020 (Murray and Lopez 1996). A Danish population-based case-control study
estimated that COPD was associated with a 16.5-fold increased risk of NTM
pulmonary disease (Andrejak et al. 2013).
The assessment of NTM-related mortality rates suffers from application of
different study approaches and interpretations. Various studies consider different
NTM disease manifestations, different NTM species, patient groups displaying
different comorbidities, varying geographic areas, and variable follow-up times
and apply different criteria for control groups (Yeung et al. 2016). We therefore
firstly focus on the results of a systematic review reporting the 5-year mortality in
patients with MAC lung disease (Diel et al. 2018), which is the most frequent cause
of pulmonary NTM infection (Johnson and Odell 2014). By reviewing the literature
until 2017, Diel et al. found a 5-year all-cause mortality of 27% with a variation
between 10% and 48%. In another study Diel et al. (Diel et al. 2017) compared the
mortality of patients with NTM pulmonary disease from Germany diagnosed in
2010 and 2011 and followed for 39 months with a control group that was matched
with respect to age, sex, and comorbidities (e.g., COPD). The mortality rate of the
patient group with NTM pulmonary disease during the 39 months was 22.4%
compared to 6% in the control group. This comparison very clearly illustrates the
high impact on mortality of NTM pulmonary infections.
The study from Diel and colleagues (Diel et al. 2017) also analyzed the costs
caused by pulmonary NTM infections in this patient cohort. The annual direct costs
plus the indirect work-loss costs attributable to NTM pulmonary disease were €
10,314.25 per patient and were about 4 times higher than for a matched control
cohort of patients with the same underlying diseases. The annual average costs in
2010 for treatment of pulmonary NTM infection in the USA were estimated to be $
9,451 and were related to the average direct costs of $ 5,113 per patient for treatment
of multidrug-resistant tuberculosis (Strollo et al. 2015). An assessment of the costs of
treatment of pulmonary NTM disease by Ballarina and colleagues (Ballarino et al.
2009) enrolling patients in 2004 and 2005 estimated the median annual cost to be $
5,772 with higher costs for M. avium compared to M. abscessus infections ($ 5,196
versus $ 11,796) and concluded that treatment of NTM pulmonary disease is
similarly expensive as treatment of MDR-TB and HIV/AIDS.
5.7 Conclusions: Prioritization of NTM Infections in Public

Health
NTM lung infections represent severe chronic and hard-to-treat diseases requiring
long-term treatments with pronounced side effects. Due to increasing numbers of
elderly and vulnerable people in the absence of prevention measures, sufficiently
informative diagnostics, and treatment options, the prevalence rates are increasing.
The mortality rates of NTM infections are substantial, and the treatment costs are
comparable to the costs for treatment of tuberculosis or HIV/AIDS. In the last years,
NTM have been established as important nosocomial pathogens causing severe

surgery-related outbreaks. Finally, patient-to-patient transmissions of M. abscessus
between CF patients have been proven. In view of these facts, we recommend giving
NTM infections a higher priority both in basic research and when deciding on public
health measures.
Note
After preparation of the manuscript, Humana Press published the book
Nontuberculous Mycobacterial Disease, edited by David E. Griffith, in the book
series “Respiratory Medicine,” which provides valuable information to specific
aspects of NTM disease.
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Tuberculosis in New Zealand: Historical
Overview to Modern Epidemiology 6
Ronan F. O’Toole
Abstract
The burden of tuberculosis in New Zealand peaked during the Second World War
with 2603 cases recorded in 1943. Control measures legislated for in the Tuber-
culosis Act of 1948 played a significant role in bringing down the national
incidence rate from over 150 cases per 100,000 to below 10 per 100,000 by the
1990s. Today, New Zealand is considered to be a low TB incidence country;
however, this designation can mask disparities in the burden of TB within the
population. The Asian ethnic group exhibits rates of TB that are approximately
50 times greater than in the European or other ethnic groups. Furthermore, among
New Zealand-born individuals, Māori experience a higher burden of TB com-
pared to their Pākehā compatriots. In this chapter, factors involved in contributing
to the risk of TB in New Zealand are examined. Data from molecular typing
studies are explored to gain an insight into the transmission of the illness in the
population. In terms of further measures to reduce the incidence of TB in
New Zealand, newer technologies that specifically detect latent TB infection
are required for pre-immigration screening from high TB burden countries.
Greater utilisation of prophylactic therapy of latent TB infection in individuals
at risk of developing TB could assist in preventing reactivated cases. And early
detection and treatment of active TB cases combined with enhanced contact
tracing would help minimise the emergence of epidemiological clusters of the
disease.
R. F. O’Toole (*)
School of Molecular Sciences, College of Science, Health and Engineering, La Trobe University,
Melbourne, VIC, Australia
Department of Clinical Microbiology, School of Medicine, Trinity College Dublin, Dublin, Ireland
School of Medicine, College of Health and Medicine, University of Tasmania, Hobart, Tasmania,
Australia
e-mail: otooler3@tcd.ie

https://doi.org/10.1007/978-981-32-9413-4_6
88 R. F. O’Toole
Keywords
Mycobacterium tuberculosis complex · MIRU-VNTR · Latent tuberculosis
infection
Abbreviations
BCG bacillus Calmette-Guérin

DHB District Health Board
EEA European Economic Area
EU European Union
ICER incremental cost-effectiveness ratio
IGRA interferon gamma release assay
LTBI latent tuberculosis infection
MIRU-VNTR mycobacterial interspersed repetitive unit-variable number tandem
repeat
MTBC Mycobacterium tuberculosis complex
NTAC National Tuberculosis Advisory Committee
NZ New Zealand
TB tuberculosis
TST tuberculin skin test
UK United Kingdom
USA United States of America
6.1 Introduction to Tuberculosis
In 2016, there were an estimated 10.4 million new cases of tuberculosis (TB) globally
corresponding to an annual incidence rate of approximately 140 per 100,000 persons
(World Health Organization 2017). Highest incidence rates of TB were seen in
South Africa (781/100,000), Lesotho (724/100,000) and the Philippines
(554/100,000) in 2016 (World Health Organization 2017). Five countries recorded
more than 0.5 million new cases nationally and accounted for >50% of the global
total in 2016, i.e. India (2,790,000 cases), Indonesia (1,020,000 cases), China
(895,000 cases), the Philippines (573,000 cases) and Pakistan (518,000 cases)
(World Health Organization 2017).
The World Health Organization (WHO) has set End TB Strategy targets to reduce
global TB mortalities by 95%, and the incidence of TB by 90% to less than 10 cases
per 100,000 by 2035 with respect to 2015 levels (World Health Organization 2014).
While industrialised countries such as New Zealand are considered to have a low TB
burden, the WHO has also established specific targets for low TB incidence
countries for the pre-elimination of TB by 2035 (corresponding to <10 TB cases
6 Tuberculosis in New Zealand: Historical Overview to Modern Epidemiology 89
per million population) and the elimination of TB by 2050 (<1 TB case per million
population) (Parish et al. 2001). Hence, an 85% drop in TB cases by 2035 and a
98.5% drop in TB cases by 2050 would be needed for New Zealand to meet
international targets. But as the incidence rate of TB in New Zealand has been
relatively stable from 2007 to 2014 at between 6 and 8 cases per 100,000 persons,
achieving this target will not be straightforward. Reaching targets for the elimination
of TB will require major advances in our understanding of the biology of the disease
as well as improvements in the effectiveness and accessibility of preventive, diag-
nostic and treatment interventions.
6.2 History of Tuberculosis in New Zealand
There has been debate stretching back many years with regard to the origin of TB in
New Zealand. A number of scholars have advocated that TB was likely brought by
Europeans to New Zealand and that there is a lack of evidence that it was present in
the indigenous Māori population prior to European colonisation (Dunsford 2008;
Brothwell and Sandison 1967; Littleton et al. 2010). For example, Littleton and
colleagues stated that ‘there is no direct evidence of its effects on Māori people until
the arrival of Europeans in the eighteenth century’ (Littleton et al. 2010). The earliest
recorded case of pulmonary tuberculosis in New Zealand dates from 1808, while
there is evidence of tuberculosis on the first voyages of the French and English to
New Zealand (Hanham 2003). The dominance of Mycobacterium tuberculosis
strains belonging to the Euro-American lineage (lineage 4) in the Māori population
would appear to correlate with the hypothesis of TB introduction with colonisation
as establishment of lineage 4 in New Zealand is unlikely to have preceded the arrival
of Europeans (Yen et al. 2013). However, one cannot rule out the possibility that TB
existed in New Zealand Māori earlier and that strain displacement took place
following introduction of the Euro-American lineage – a scenario that has been
suggested for TB in relation to the post Columbian migration of Europeans to the
Americas (Gagneux 2012). In line with this, there are also scholars who have
proposed that TB was present in New Zealand prior to colonisation. In particular,
Te Rangi Hiroa in his medical thesis, published in 1910, stated ‘That tuberculosis
existed among Māoris before the advent of Europeans, I feel certain’ (Te Rangi
Hiroa 1910). More investigations and experimental data are needed to verify a
precise timeline for the emergence of TB in New Zealand. But what is clear is that
Māori were disproportionately affected by TB in the years following colonisation of
New Zealand. As noted by Te Rangi Hiroa, in relation to phthisis (tuberculosis):
The change in environment has allowed phthisis to make great headway. The crowding
together in single rooms aids its spread. There is no doubt that the lapse in the sanitary
regulations and conditions, with the change in clothing, food and work, brought about by the
first contact with the Europeans, made the conditions more favourable for tuberculosis,
which was kept in check by the open-air active life of the old-time Māori. The privations and
starvation owing to insufficient food cultivated which followed the waikato war helped to
90 R. F. O’Toole
disseminate the seeds of disease in that district until it became very widespread (Te Rangi
Hiroa 1910).
The burden of TB in New Zealand increased until reaching a peak in 1943 during
the Second World War of 2603 cases corresponding to a national rate of 159.1 cases
per 100,000 persons (Das et al. 2006). Māori continued to experience higher rates of
TB than their European counterparts. At the height of TB incidence in New Zealand,
TB mortality in Māori was 422 per 100,000 as opposed to 39 per 100,000 in
non-Māori in 1942 (Grant 2011). The Tuberculosis Act, which was introduced in
New Zealand in 1948, sanctioned a number of provisions for the ‘treatment, care,
and assistance of persons suffering or having suffered from tuberculosis, and for
preventing the spread of tuberculosis’ (New Zealand Parliament 1948). The
measures included the duty of medical practitioners to notify TB cases; powers for
medical officers of health to ensure that TB patients obtain treatment; direct
precautions to prevent further spread such as patient isolation, vocational training
and industrial rehabilitation courses for patients; and a compensation scheme for
healthcare workers believed to have contracted the disease in the course of their
employment (New Zealand Parliament 1948). From the late 1940s onwards, the TB
incidence rate in New Zealand steadily declined until it began to plateau at approxi-
mately 10 cases per 100,000 in the 1995–2004 period (Das et al. 2006). The post-
Second World War decline in tuberculosis incidence in New Zealand, and in other
industrialised countries such as the UK, can be attributed not only to the
strengthening and coordination of control measures but also to improvements in
socioeconomic development accompanied by better nutrition and living and work-
ing conditions (Glaziou et al. 2018).
6.3 Current Epidemiology of Tuberculosis in New Zealand
Today, New Zealand is regarded as a low TB incidence country with

302 notifications including 290 new cases and 12 relapse/reactivation cases in
2014 (incidence rate of 6.7 cases per 100,000 population) (Institute of Environmen-
tal Science and Research Ltd. (ESR) 2015). However, the distribution of the disease
is not uniform across the population with some at-risk groups exhibiting disparate
TB rates. For example, the notification rate of TB in the Asian ethnic group was 56.8
times higher (34.1 per 100,000) than in the European or other ethnic groups (0.6 per
100,000) in 2014 (Institute of Environmental Science and Research Ltd. (ESR)
2015). Furthermore, higher incidence rates are reported for Māori (5.3 per
100,000) and Pacific peoples (16.9 per 100,000) than for European or others.
Geographically, disparities also exist in the distribution of TB. In 2014, the
highest rates were reported in the District Health Board (DHB) regions of Auckland
(14.6 per 100,000, 69 cases) followed by Capital and Coast (11.8 per 100,000,
35 cases) and Counties Manukau (9.4 per 100,000, 48 cases), with lowest rates in
Waitemata DHB region (6.4 per 100,000, 36 cases) (Institute of Environmental
Science and Research Ltd. (ESR) 2015). Intermittent outbreaks of TB can signifi-
cantly impact the regional rates from year to year.
New Zealand TB notifications reported to the national notifiable diseases data-
base (EpiSurv) by the country’s four reference mycobacteriology laboratories
located in Auckland, Hamilton, Wellington and Christchurch have high levels of
data completeness. This includes data on patient risk factors for the development of
TB. The most commonly reported TB risk factors have been birth outside
New Zealand (76.6% of cases with risk factor data) and current or recent residence
with person(s) born outside New Zealand (76.3%) (Bissielo et al. Institute of
Environmental Science and Research Ltd.; Institute of Environmental Science and
Research Ltd. (ESR) 2015). These two risk factors have dominated TB cases in
New Zealand over several years. Notification data from 2005 to 2009 showed that
70.1% of new TB cases occurred in persons born overseas (Ministry of Health
New Zealand 2010). Patient age is also associated with TB with highest rates
observed in the 15–39 and 60 age groups. The latter 60 age group experiences
the highest rate of hospitalisations (43/58, 74.1% of cases). Furthermore, all four
recorded fatalities from TB in 2014 occurred in the 60 age group (Institute of
Environmental Science and Research Ltd. (ESR) 2015).
Among New Zealand-born individuals, Māori make up 15% of New Zealand’s
population but 51.5% of its TB cases, compared to 74% of the population being of
European descent and 16.2% of TB cases recorded in the European or other ethnic
groups (Institute of Environmental Science and Research Ltd. (ESR) 2015; Stats NZ
2013). This is a common trend seen in other jurisdictions where significant disparity
exists between the indigenous and nonindigenous populations with respect to the
burden of TB. Among indigenous populations in the USA, a 5.4-fold higher rate of
TB has been reported in American Indians and Alaskan Natives compared to
non-Hispanic Caucasians (Tollefson et al. 2013). The TB incidence rate in the
Aboriginal population has been recorded at 5.3- and 12.9-fold higher in Australia
and Canada, respectively, relative to the domestic-born non-Aboriginal population
(Barry et al. 2012; Public Health Agency of Canada 2012).
With regard to drug resistance, there were three (1.2%) cases of multidrug-
resistant (MDR) TB, defined as resistance to at least isoniazid and rifampicin, and
an additional two cases that were mono-resistant to rifampicin, in New Zealand in
2014 (Institute of Environmental Science and Research Ltd. (ESR) 2015). One of the
MDR-TB cases is believed to have developed during antituberculous drug treatment
undertaken in New Zealand (Institute of Environmental Science and Research Ltd.
(ESR) 2015). Although this may appear to be a relatively small number of cases,
MDR-TB is more difficult and costly to treat. Globally, the treatment completion
success rate drops from 83% overall for TB to 54% for MDR-TB cases due to
treatment failure, loss to follow-up and death of patients (World Health Organization
2017). In addition, the estimated costs for treating TB (USD $17,000 in the USA,
€10,282 in 15 European Union (EU) countries, per drug-susceptible TB case)
increase substantially with multidrug-resistant forms of TB (USD $134,000 in the
USA, €57,213 in 15 EU countries, per case) (Diel et al. 2014; Bhakta et al. 2004).
92 R. F. O’Toole
Therefore, only a small number of MDR-TB cases are required to place an additional
financial strain on healthcare resources.
Regarding site of infection, of the 290 new TB cases notified in New Zealand in
2014, 179 cases (61.7%) had pulmonary disease, including 55 cases (19.0%) which
also had extrapulmonary involvement. An additional 111 cases (38.3%) were
reported as being exclusively extrapulmonary TB (Institute of Environmental Sci-
ence and Research Ltd. (ESR) 2015). The most common extrapulmonary TB site of
infection recorded was a lymph node (excluding abdominal), followed by pleural
and intra-abdominal sites (excluding renal) (Institute of Environmental Science and
Research Ltd. (ESR) 2015). There are detectable differences in the clinical
characteristics of cases born in New Zealand with respect to overseas-born cases.
Pulmonary disease was reported in approximately 75% of New Zealand-born TB
cases between 2010 and 2014. In contrast, the level of pulmonary disease was lower,
approximately 53%–57.5% between 2010 and 2014, in new TB cases in patients
who were born outside New Zealand (Institute of Environmental Science and
Research Ltd. (ESR) 2015).
6.4 Molecular Epidemiology of Tuberculosis in New Zealand
To better understand the biology of TB in New Zealand, it is necessary to examine

the genetic make-up of the strains circulating in the population. One of the first
nationwide studies on the molecular typing of M. tuberculosis complex in
New Zealand was published in 2008 (Sexton et al. 2007). Here, clinical isolates
from 2003 to 2007 were typed using the previously described mycobacterial
interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing
method (Supply et al. 2006). Of 1736 notifications in New Zealand for TB disease
caused by species M. tuberculosis during the above time frame, 1328 cases were
matched to available MIRU-VNTR data, 94.4% of which were new cases and 5.6%
were due to relapse or reactivation (Sexton et al. 2007). A total of 866 cases (65.2%)
had a unique MIRU-VNTR genotype and, therefore, were classified as non-cluster
cases. On the other hand, 462 cases (34.8%) had a non-unique MIRU-VNTR
genotype, which was suggestive of potential clustering of these TB cases (Sexton
et al. 2007). 140 different MIRU-VNTR genotypes made up the clusters. The
so-called Rangipo genotype was associated with the largest cluster size of 57 cases
and with the most number of DHBs, i.e. 13 out of 21 then DHBs compared to other
clusters which were associated with a median of 2 DHBs (Sexton et al. 2007). This
genotype had previously been linked to a large outbreak of TB involving 61 cases
which occurred in New Zealand between November 1996 and May 2000 (De Zoysa
et al. 2001). A subsequent outbreak due to the Rangipo genotype occurred in
Hawke’s Bay in 2002 and entailed 19 active cases (McElnay et al. 2004). Next-
generation sequencing studies are beginning to shed light on factors that may
contribute to the relatively high prevalence of the Rangipo strain among cluster
and outbreak cases (Colangeli et al. 2014; Gautam et al. 2017; Mac Aogain et al.
2016).
The cluster cases from 2003 to 2007 were more likely to be less than 20 years of
age, to be of Māori or Pacific ethnicity and to reside in the Northland, Counties
Manukau, Taranaki, Hawke’s Bay, Whanganui or Hutt Valley DHB regions (Sexton
et al. 2007). With four clusters involving patient numbers >20 cases each, this was
strongly indicative of transmission among TB contacts. Indeed, one of the main TB
risk factors associated with cluster cases was contact with a confirmed case of TB,
followed by birth in New Zealand or a Pacific Island country, and the pulmonary
form of TB. Non-cluster cases in contrast were more likely to be aged between
30 and 39 years or > 70 years, to be of Asian ethnicity and to reside in the Auckland
or Nelson Marlborough DHBs. This indicates that a distinct set of contributory
factors exist for TB clusters in New Zealand compared with unique cases.
It is important to note that M. tuberculosis is not a single organism but instead is a
complex of seven distinct phylogenetic lineages to date based on genomic
polymorphisms (Brites and Gagneux 2017; Comas et al. 2013). A number of studies
have found that specific lineages within the M. tuberculosis complex (MTBC) differ
with respect to geographic distribution, transmissibility, site of infection, virulence,
immunogenicity and prevalence of drug resistance (Reed et al. 2009; Gagneux and
Small 2007; Gagneux et al. 2006; Click et al. 2012; Nahid et al. 2010; Lan et al.
2003; Parwati et al. 2010; Borrell and Gagneux 2009; Niemann et al. 2010; National
Institute for Public Health and the Environment 2006). A study by Yen and
colleagues examined the relative abundance of the major M. tuberculosis lineages
in New Zealand (Yen et al. 2013). They found that the predominant lineage in
New Zealand was lineage 4 (Euro-American) constituting 37.8% of isolates in
2010–2011, followed by lineage 1 (Indo-Oceanic, 22.6%), lineage 2 (East Asian,
19.5%) and lineage 3 (East African Indian, 17.7%) (Yen et al. 2013). Lineage 2 was
associated with multidrug resistance in New Zealand, in agreement with studies in
other jurisdictions (National Institute for Public Health and the Environment 2006;
Niemann et al. 2010), and accounted for 58.1% of MDR-TB cases between 2002
and 2011.
The investigators also compared the epidemiology of the MTBC lineages to
determine whether any patterns emerged with respect to the country of origin and
ethnicity of TB patients. Here, they found that in the New Zealand-born population,
lineage 4 (Euro-American) was the dominant lineage among both the NZ European
and Māori populations (82.6% and 64.4%, respectively) (Yen et al. 2013). Lineage
3 was significantly more prevalent in Māori than in NZ Europeans with no cases in
the latter group in 2010 and 2011. The reasons behind this observation are not
currently known.
Among migrant TB cases, the predominant lineages corresponded to MTBC
lineages that are prevalent in their respective countries of origin (Yen et al. 2013).
The significance of this finding is discussed in the next section. No significant
relationships with respect to patient variables age or gender were detected with
regard to MTBC lineages 1–4 over the years of the study; however, males had a
higher association with M. bovis infection than females for New Zealand isolates
from 2006 to 2010 which may reflect differences in relation to exposure risk during
the period analysed (Yen et al. 2013).
94 R. F. O’Toole
6.5 Measures to Reduce the Incidence of Tuberculosis in

New Zealand
In New Zealand, >70% of cases occur in overseas-born individuals. A trend that has
been seen in many low TB burden countries is decreasing TB in the native-born
population and increasing TB in the migrant population as a proportion of total cases
(Hanway et al. 2016). Therefore, reducing the incidence of TB in the migrant
population is key to bringing down the national rate of TB in New Zealand.
Internationally, it is believed that most TB in the foreign-born population in
industrialised countries occurs due to reactivation of latent TB infection (LTBI)
rather than manifesting as a continuation of an existing case of active TB (White and
Houben 2014; Ricks et al. 2011). This includes New Zealand’s nearest neighbour,
Australia, whose National Tuberculosis Advisory Committee (NTAC) recently
stated that ‘In 2013, 88% of 1322 notifications in Australia were in the overseas-
born population (incidence 19.5 per 100,000 v. 1.0 per 100,000), with this propor-
tion rising over the course of the last decade. Combined with epidemiological
evidence of low local transmission, this strongly implies that the vast majority
resulted from reactivation of latent infection acquired prior to immigration’ (Stock
and National Tuberculosis Advisory Committee (NTAC) 2017). Molecular
genotyping studies conducted in a number of countries, including New Zealand,
have identified a strong association between the genetic lineage of the MTBC strain
causing TB in a migrant patient and the predominant MTBC lineage in the patient’s
country of origin (Yen et al. 2013; Filliol et al. 2006; Reed et al. 2009). Therefore,
for a large proportion of overseas-born cases, initial infection with M. tuberculosis
occurs prior to entry into New Zealand (O’Toole and Freeman 2013).
In New Zealand, confirmation of TB status in pre-immigration screening is based
on a chest x-ray (New Zealand Immigration 2018b). Citizens of countries that are not
on New Zealand Immigration’s list of low TB incidence countries are required to
undergo a chest x-ray if applying for a visa to stay in New Zealand for longer than
6 months (New Zealand Immigration 2018a). However, the chest x-ray is primarily
intended to detect active pulmonary TB and has limited sensitivity in the detection of
LTBI. Therefore, many cases of LTBI in migrants will be undetected with the
currently used TB diagnostic test in pre-immigration screening. This problem is
not unique to New Zealand, but as recently stated by the Australian NTAC, ‘Given
the very low rates of transmission within Australia, it is clear that progressing toward
TB elimination is largely contingent on the implementation of strategies to detect
and treat LTBI in migrants from high incidence countries’ (Stock and National
Tuberculosis Advisory Committee (NTAC) 2017).
One of the limitations with screening for LTBI has been the cross-reaction
between the tuberculin skin test (TST) and previous vaccination with BCG, leading
to a high proportion of migrants from high TB burden countries testing positive in
the TST (Wells et al. 1997; Orlando et al. 2010). However, newer diagnostic tests
based on the interferon-gamma release assay (IGRA) target non-BCG antigens,
ESAT-6 and CFP-10, and, therefore, deliver greater specificity for latent TB
(Lalvani et al. 2001; Lalvani 2003). While screening all migrants for LTBI is not
economical, one study of 213 immigrants found that screening of migrants from
countries with a TB incidence rate of greater than 150/100,000 with the
QuantiFERON-TB Gold In-tube IGRA test detected 49–71% of LTBI cases with
an incremental cost-effectiveness ratio (ICER) of GBP £31,867.10 per case averted
(Pareek et al. 2013). Another study of 1229 immigrants reported that screening of
immigrants (from countries with a > 150/100,000 TB incidence rate) specifically for
LTBI with IGRA testing identified 92% of infected immigrants and prevented
29 additional TB cases at an ICER of GBP £20,819 per additional case averted
(Pareek et al. 2011). The investigators concluded that ‘Screening for latent infection
can be implemented cost-effectively at a level of incidence that identifies most
immigrants with latent tuberculosis, thereby preventing substantial numbers of
future cases of active tuberculosis’ (Pareek et al. 2011).
While further consideration will need to be devoted to the prevention and
management of TB in the migrant population in New Zealand, it is important to
note that a number of studies have established that immigrants are not a major source
of TB infection for the native-born population (Barniol et al. 2009; Kamper-
Jørgensen et al. 2012; Gautam et al. 2018). In Europe, a systematic review concluded
that ‘TB in a foreign-born population does not have a significant influence on TB in
the native population in EU/EEA’ (Sandgren et al. 2014). Therefore, it is important
to maintain the focus on New Zealand’s obligations as a ‘global citizen’ to assist in
reducing the burden of TB internationally and in helping migrants to New Zealand
from high TB incidence countries by minimising their risk of developing TB.
In terms of addressing TB in the New Zealand-born population, as noted above,
Māori experience an approximate eightfold higher incidence rate of TB compared to
the European or other groups and make up more than 50% of TB cases among New
Zealand-born individuals (Institute of Environmental Science and Research Ltd.
(ESR) 2015). Recent initiatives to overcome this disparity in TB rates include the
announcement in 2018 by the Health Research Council of New Zealand of funding
for a feasibility study to characterise the reservoir of LTBI among Māori and enable
strategic intervention with preventive treatment to be considered (Health Research
Council of New Zealand 2018).
6.6 Conclusions
New Zealand is regarded as a low TB incidence country with approximately

300 cases each year. Nevertheless, particular populations in New Zealand experience
disproportionately high levels of TB. Furthermore, the country’s national incidence
rate must be reduced substantially in order to meet TB eradication targets for 2035
and 2050 that have been set by the World Health Organization. As annual TB rates
among New Zealanders of European descent have dropped below 1 case per
100,000, reducing the burden of TB in New Zealand will require investment of
healthcare resources towards groups who are at a higher risk of developing the
illness. Measures to improve the detection of active TB may reduce diagnostic and
treatment delays and hence minimise transmission and epidemiological clusters of
96 R. F. O’Toole
TB. In addition, where appropriate, further screening and preventive therapy

for latent TB infection may assist in decreasing the numbers of reactivated cases.
Competing Interests The author declares that he has no financial or other conflicts of interest.
Ethical Approval Not required.
Funding Not applicable.
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Part II
Molecular and Infection Biology of
M. tuberculosis
Mycobacterial Methyltransferases:
Significance in Pathogenesis and Virulence 7
Sonam Grover, Rishabh Gangwar, Salma Jamal, Sabeeha Ali,
Khairun Nisaa, Nasreen Z. Ehtesham, and Seyed Ehtesham Hasnain
Abstract
Mycobacterium tuberculosis (M.tb) is a pathogen of incredible international
prominence owing to its persistence for long duration inside human host in
both active and latent form, complex eradication methods and imposing long-
term treatment procedures. The mechanisms employed by M.tb to adjust and
survive inside extreme host environment and to evade the immune system of host
need to be explored in greater depth in order to enable the rational design of novel
treatment strategies. Methylation of biomolecules plays a significant role in
almost every kingdom of life but has not been extensively addressed in the case
of M.tb. The genome of M.tb codes for 121 methyltransferases (MTases) in spite
of the reductive evolution of its genome. In the present chapter, we will discuss in
detail about various MTases modifying DNA, RNA, protein, mycolic acid and
S. Grover · R. Gangwar · S. Jamal

S. Ali
Kusuma School of Biological Sciences, Indian Institute of Technology, New Delhi, Delhi, India
K. Nisaa
Department of Life Sciences, Ben Gurion University of the Negev, Beersheba, Israel
N. Z. Ehtesham
Inflammation Biology and Cell Signaling Laboratory, ICMR-National Institute of Pathology,
Safdarjung Hospital Campus, New Delhi, Delhi, India
e-mail: nasreenzehtesham.nip@gov.in
S. E. Hasnain (*)
Dr Reddy’s Institute of Life Sciences, University of Hyderabad Campus, Hyderabad, India
e-mail: vc@jamiahamdard.ac.in

https://doi.org/10.1007/978-981-32-9413-4_7
104 S. Grover et al.
other biomolecules of M.tb along with the host. This will also shed light on how
methylation is implicated in virulence and influences the mechanism of patho-
genesis of M.tb.
Keywords
Methyltransferases · Mycolic acid · Epigenetic regulation · Drug resistance
7.1 Introduction
Mycobacterium tuberculosis (M.tb), responsible for causing tuberculosis (TB), is a

micro-organism that is posing greatest threats to humanity. According to the World
Health Organization (WHO) report, around one-third of the population is exposed
worldwide, and the 5% amongst the exposed ultimately develop disease that
translates into eight million new TB cases per year (WHO 2017). The process of
M.tb infection can be divided into a number of stages (Cooper 2009). The disease
starts when the microbes are breathed out by infected individual which are further
transferred by air droplets. These bacteria are then inhaled by healthy individuals
which is the primary stage of infection during which the bacteria inhabit
macrophages. Macrophages are large phagocytes that serve at the forefront of host
immune defence against pathogenic microbes. These are also the primary sites of M.
tb habitation contrasting other bacteria that need to avoid the process of phagocytosis
for survival (Warner and Mizrahi 2007). M.tb preferentially targets macrophage
vacuoles where they are constantly exposed to the macrophage’s antimicrobial
effectors that include reactive oxygen plus nitrogen species as well as low pH that
could compromise the primary infection, latency and reinfection or kill M.tb (Gorna
Alina et al. 2010). Hence, in order to maintain its viability and virulence, it is
compulsory for M.tb to have effective systems to counteract the impact of such
assaults generated by the environment and the host (Mizrahi and Andersen 1998;
Warner and Mizrahi 2007; Dos Vultos et al. 2009; Kurthkoti and Varshney 2012;
Voskuil et al. 2011).
Biology relies upon the enlarged repertoire of interactions that occur when
biomolecules such as proteins, DNA or RNA are modified. The process of methyla-
tion of nucleic acid and protein expands the range of functions presented to their
modified substrates and plays different roles in cellular signalling and regulation of
macromolecular function. From current scientific research, it has become evident
that methylation stands beside phosphorylation as a major regulating factor in the
functioning of proteins. Methylation of DNA causes epigenetic changes in the
genome, thus affecting gene expression, replication and chromatin remodelling. In
the similar way, RNA methylation provides integrity and structural stability to
ribosomes at the time of translation. These modifications increase multitudes of
chemical interactions that play roles in variety of cellular mechanisms (Clarke and
Tamanoi 2006). Thus, methylation controls numerous cellular processes of the
organism according to the metabolic status.
7 Mycobacterial Methyltransferases: Significance in Pathogenesis and Virulence 105
Fig. 7.1 Overview of methyltransferase reaction and fate of S-adenosylhomocysteine (SAH)
The process of methylation is catalysed by methyltransferases, during which a

methyl group is transferred from S-adenosylmethionine (SAM, a methionine deriv-
ative) to nucleic acids, proteins, lipids and secondary metabolites. In methylation
reaction, SAM is altered resulting in the formation of S-adenosylhomocysteine
(SAH), which can then be broken down to methionine and cysteine which are
sulphur-containing amino acids or can be further metabolized to the antioxidant
glutathione (Fig. 7.1). Thus, methylation directly feeds into sulphur metabolism and
oxidative balance. More than 1% of genes have been predicted to encode
methyltransferases in the mammalian genome (Katz 2003), while in the case of M.
tb, this number has increased to 3% in spite of genome reduction of M.tb. The large
number of MTases is able to draw the attention of scientists to explore their role in
pathogenicity and virulence.
7.2 Types of Methyltransferase
In M.tb proteome, about 121 methyltransferases (MTases) have been identified

(Grover et al. 2016), which employ various substrates that include DNA, RNA,
intermediates of mycolic acid biosynthesis, protein and other fatty acids. These
MTases can be further divided into five main categories in case of M.tb: mycolic
acid MTases, DNA MTases, RNA MTases, protein MTases and other MTases.
7.2.1 Mycolic Acid Methyltransferases
Mycolic acids (MAs) which are the 2-alkyl, 3-hydroxy long-chain fatty acids (FAs)
form a distinctive feature of the thick lipid-rich membrane of M.tb and other related
mycobacterium (Daffé and Draper 1998; Barry et al. 1998). MAs are very-long-
chain (C60–C90) fatty acids, having an alkyl side chain at the α-position and a
hydroxyl group at the β-position (Fig. 7.2a), either unbound or bound to the terminal
penta-arabinofuranosyl units of arabinogalactan (AG) that, along with
Fig. 7.2 (a) (i) Fundamental structure of a mycolic acid shown using M.tb α-mycolic acid as an
example; (ii) structure of methoxy mycolic acid subclass from M.tb; (iii) structure of keto mycolic
acid subclass from M.tb (Nataraj et al. 2015). (b) Modifications of the mycolic acid mero-chains
and involvement of MTases: structures of the mero-chains of representative types of MAs from
mycobacteria. The configuration of double bonds and cyclopropanes is either cis or trans (with an
adjacent methyl branch). The established functions of the MTases are indicated
peptidoglycan, forms the insoluble cell wall skeleton (Daffé and Draper 1998;
McNeil et al. 1991). Recent electron microscopy visualization revealed that the
unbound and bound forms presumptively play a vital role within the exceptional
architecture as well as the impermeable cell envelope, participating within the two
leaflets of the outer membrane of the mycobacterium, additionally referred to as
mycomembrane (Hoffmann et al. 2008; Sani et al. 2010; Zuber et al. 2008).
The mycomembrane serves as a physiological blockade between the bacteria and

its environment and has a significant role in each persistence and virulence. MAs
present in mycomembrane are responsible for not only the relative cell wall imper-
meability, specifically to antibacterial agents and drugs, but also mycobacterial acid
fastness (Laval et al. 2008; Bhatt et al. 2007). It has been widely established in slow-
growing mycobacteria responsible for pathogenesis that MAs are modified by
addition of methyl branches, cyclopropane rings, methoxy groups and ketones to
generate a series of three main mycolic acids: α-mycolates (two cis-cyclopropane
rings), ketomycolates (a single cis or trans form of cyclopropane ring and a ketone
group) and methoxymycolates (a single cis- or trans-cyclopropane ring and a
methoxy group). Structural diversity in MAs is generated by the modification of
unsaturated meromycolic acid, catalysed by a family of AdoMet-dependent MTases,
to generate cis and trans forms of cyclopropanes and several other mycolates (Barry
et al. 1998). So far, eight distinctive MTases have been characterized, namely,
CmaA1 (Rv3392c), CmaA2 (Rv0503c), MmaA1 (Rv0645c), MmaA2 (Rv0644c),
MmaA3 (Rv0643c), MmaA4 (Rv0642c), PcaA (formerly UmaA2; Rv0470c) and
UmaA (Rv0469), which are responsible for certain modifications of mycolic acid
moieties of the cell envelope (Fig. 7.2b).
The growth of Mycobacterium, in presence of the labelled methionine, points out
that the methyl group of methionine can be directly integrated into MAs, and the
bridging methylenes of the methyl branches adjacent to methoxy, trans-olefins and
keto moieties, the carbon of the methoxy functionality and also the cyclopropane
rings are all derivatives of methionine, presumptively via SAM (Daffé et al. 1991;
Lacave et al. 1987; Danielson and Gray 1982).
The first direct evidence of the long proposed idea that the meromycolyl groups of
cyclopropane were gained from the double bonds (Lederer 1969; Grogan and
Cronan 1997) came with the identification of cyclopropane mycolic acid synthase
(Cmas1), product of cma1 gene from M.tb that enables M. smegmatis to produce
cyclopropane-containing MAs in large quantities (Yuan et al. 1995). This gene has
shown strong homology within the region known to bind SAM of other
SAM-dependent methyltransferases and was also found to have 34% similarity
with the cyclopropane fatty acid synthase (Cfas) of E. coli (Grogan and Cronan
1997; Wang et al. 1992).
Another gene from M.tb, cma2, was discovered on the basis of cma1 sequence.
Categorization of the purified chimeric mycolates is obtained as a consequence of
heterologous expression of Cmas2, the gene product of cma2 in M. smegmatis,
shown to attach a cyclopropane ring at the proximal position (George et al. 1995).
Interestingly, Cmas2 has the specificity for cis double bonds as it cyclopropanates
the α1-mycolates and not the α2 from M. smegmatis. Although both cis and trans
forms of cyclopropane-containing mycolates are produced by M.tb (Minnikin and
Polgar 1967), only cis-cyclopropanes are produced by both cma1 and cma2. The
existence of trans-cyclopropanes suggests the existence of no less than one more
enzyme in M.tb, capable of introducing such modification in MAs. The trans-
cyclopropanes always go together with a nearby methyl branch, suggesting the
involvement of two counterparts of SAM in their formation; the first forms a
trans-olefin with a nearby methyl group and the second from the actual cyclopropane
ring. Indeed, two additional homologs of cma1 and cma2 which might encode such
activities have been revealed through the analysis of the complete M.tb genomic
sequence.
Remarkably, a group of four additional genes was identified by DNA

hybridization to a cma1 probe in M.tb (Yuan and Barry 1996). In addition to cma1
and cma2, similarity was found amongst all the four genes. By analysing the
resultant mycolates following the introduction of these genes, either alone or in
combination, into M. smegmatis, it was shown that mma4 gene product adds a
hydroxyl group along with a nearby methyl branch at the distal position, whereas
the mma3 encodes an enzyme that methylates this hydroxyl resulting in the forma-
tion of a methyl ether. The new mycolate species were just like the ketomycolate and
methoxymycolate series from M.tb (Yuan and Barry 1996). Interestingly, mma2
encodes a cyclopropanation enzyme that is like cma2 which alters the proximal
position. The purpose for this obvious duplication of function is uncertain. One
theory is that Cmas2 cyclopropanates α-series, whereas Mmas2 cyclopropanates the
oxygenated series of mycolates. The heterologous expression of mma1 seemed to
have no consequence in M. smegmatis; however, when mma1 was overexpressed in
M.tb, a significant increase in the quantities of trans substituents has been observed
at the proximal position (Yuan et al. 1997). Strain overexpressing mma1, showing
the presence of trans form of cyclopropanes and trans double bonds, with nearby
methyl branches, has led to the inference that Mmas1 transforms the proximal cis
double bond to a trans double bond with an allylic methyl branch. The endogenous
enzyme occurring in limiting quantities in M.tb would have catalysed trans-cyclo-
propane formation. A recent study reported these enzymes to be removed by
homologous recombination, and a feeble immune response of the host as well as a
substantial attenuation of M.tb was observed (Barkan et al. 2012).
While looking for the presence of these MTases in other mycobacterium species
through BLAST analysis, it was found that UfaA (Rv0447), having cyclopropane-
fatty-acyl-phospholipid synthase activity, is limited to true pathogens only and is
lacking in all the non-pathogenic and opportunistic mycobacterium species. UfaA
was revealed to catalyse tuberculostearic acid (10-methylstearic-acid, TSA) biosyn-
thesis. TSA is a clinical marker of the disease which makes up a major lipid moiety
of cell wall of the mycobacteria (Meena and Kolattukudy 2013). The transfer of the
methyl group from SAM to the double bond of oleic acid in phosphatidylcholine or
phosphatidylethanolamine to produce TSA is catalysed by UfaA (Meena and
Kolattukudy 2013). The aforementioned results indicated that a specific kind of
methylation is involved in immune response modulation and pathogenicity.
7.2.1.1 Mycolic Acid Methyltransferases’ Relation to Virulence

The role of modification of MA in pathogenesis of M.tb is only moderately under-
stood. Many of the genes responsible for MA modification are conserved amongst
slow-growing pathogenic mycobacteria (such as M. leprae, M. marinum and other
members of the M.tb complex) which indicates towards the role of these genes in
pathogenesis. Overall, expression of these enzymes resulted to the synthesis of
various subfamilies for which the studies delineated below have found remarkable
and exact roles of these enzymes despite of very subtle differences in their structures.
The studies on MAMTs which were lacking mutants in mice provided proof for the
significance of modifications of MA in pathogenesis. It was demonstrated by
Dubnau et al. that inactivation of hma (known as cmaA also and recently as mmaA4)
gene in M.tb leads to an extreme modification in the permeability of its envelope
together with the loss of oxygenated MAs. When this mutant was tested in an
infected mouse model by aerosolization, it exhibited an attenuated phenotype during
the first 3 weeks of infection, indicating that the oxygenated MAs are essential in the
infection process (Dubnau et al. 2000). Importantly, this study pointed towards the
precise roles of each MAMTs in the biogenesis of MAs and their part in virulence. In
the continuation of this line of research, Glickman et al. revealed the loss of cording
by the deletion of pcaA gene, which encodes for an enzyme catalysing the proximal
cyclopropanation of α-mycolates. This ΔpcaA M.tb strain (Glickman et al. 2000) has
several phenotypes: defective persistence, defective growth in the first week of
mouse infection (Rao et al. 2005) and unable to kill infected mice (Glickman et al.
2000). These observations indicated that the site-specific cyclopropanation of MAs
could be an imperative determining factor of the M.tb and host interaction. Glickman
et al. have demonstrated that an M.tb ΔcmaA2 strain lacked the trans ring on the
methoxy- and ketomycolates (Glickman et al. 2001). This mutant was also found to
be overvirulent in mice, prompting an exaggerated immune response which leads to
extreme tissue damage (Rao et al. 2006). Reconstituted methoxymycolate produc-
tion was shown by mmaA3-complemented M. bovis BCG SSI as compared to wild
type, which lacks functional MmaA3 protein. This mutant was also found to be
attenuated (Belley et al. 2004).
The investigation of the vitality of each MAMT and deletion of the seven
MAMTs existing in M.tb exhibited that none of them is as such essential and that
all of them may be deleted in spite of the fact that a certain sequence in the deletion
must be followed to permit cellular adjustment of permeability of the cell and
consequently survival. The viable strain generated through this technically challeng-
ing sequential deletion of MAMTs was incapable of making cyclopropanated or
oxygenated MAs, which resulted in massive changes in cell appearance such as
susceptibility to detergents and total loss of acid fastness. Interestingly, it has been
also shown that the total absence of cyclopropanation leads to severe reduction
during the first week, whereas complete deletion of MAMTs confers attenuation in
the second week of infection after aerosol infection in the mouse. This strain also
generated a significant hyperinflammatory response from the host (Barkan et al.
2012). Thus, there are substantial evidences of immunomodulatory role of the MA
modification, and MAMTs have many explicit functions in M.tb pathogenesis,
which reinforces this enzyme class as an interesting target for drug development
against mycobacteria.
7.2.2 DNA Methyltransferases
An interesting point to discuss is how M.tb intelligently sustains its gene expression
profile. M.tb genome is rich in GC content (~ 65%). In the case of eukaryotes, a
number of mechanisms are explored that heritably regulate expression patterns, but
prokaryotes have DNA methylation as the only known mechanism to attain
epigenetic inheritance. DNA could be methylated at adenine and cytosine residues,

forming N6-methyladenine as well as N4-methylcytosine and 5-methylcytosine. In
higher eukaryotes, cytosine methylation is validated as a significant regulatory
mechanism of gene expression, but recent reports have proposed that methylation
of cytosine also plays pivotal roles in prokaryotic gene expression (Militello et al.
2012; Kahramanoglou et al. 2012), although in prokaryotes N6-methylation of
adenine is a well-established mechanism of gene expression via epigenetic regula-
tion (Casadesus and Low 2006; Campbell and Kleckner 1990).
DNA MTases act by transferring the methyl group from SAM to specific residues
in double-stranded DNA. Prokaryotic methylation can be regulated either as a part of
host modification or in an independent manner such as by deoxyadenosine methyl-
ase (Dam) or deoxycytosine MTase (Dcm). In Dam-dependent methylation, GATC
locus is recognized, and methylation of adenine occurs at N6 position (Marinus
1987), while in Dcm-dependent methylation, CCA/TGC locus is recognized and
methylates the internal cytosine at the C5 position (Schlagman et al. 1976).
Total absence of both Dam and Dcm MTase activities in different strains of M.tb
including H37Ra, H37Rv and M. smegmatis was reported (Hemavathy and Nagaraja
1995). However, analysis of genomic DNA from M.tb and M. smegmatis was shown
to contain significant amount of 6-methyladenine and 5-methylcytosine indicating
the presence of DNA MTase other than Dam or Dcm. Since then, a variety of
MTases have been reported in M.tb (Srivastava et al. 1981). So far in M.tb, there are
121 characterized and hypothetical MTases reported in its genome (Grover et al.
2016). Out of 61 characterized MTases, 5 are characterized to be DNA MTases.
7.2.2.1 DNA Methylation Involved in Damage-Induced Repair

M.tb is an intracellular pathogen that persists and survives inside its host, i.e. human
macrophages. Consequently, it is exposed to many unfavourable surroundings such
as alkylating stress and exposure to oxygen as well as nitrogen radicals which can
cause harmful effects comprising DNA damage. Cells have adopted numerous DNA
repair pathways to counter the mutagenic/cytotoxic effect of methylation on DNA. A
study by Yang et al. demonstrated two co-operonic DNA MTases in ada operon of
M.tb which are involved in inducible repair of DNA alkylation damage. O6-
Methylguanine is one of the major mutagenic lesions removed by MTases under
ada operon, a very well-characterized mechanism in E. coli. The M.tb ada operon
encodes a fused protein made up of AdaA and AlkA (Rv1317c) in addition to a
separate AdaB/OGT (Rv1316c) protein, both of which possess DNA MTase activ-
ity. The study suggested that AdaB neutralizes the mutagenic influence of O6-
methylation of guanine, whereas AdaA-AlkA by its MTase activity removes methyl
groups from innocuous methylphosphotriesters in DNA (Yang et al. 2011).
Another detailed study on MtOGT (O6-methylguanine MTase; Rv1316c)
characterized its potential to ensure M.tb survival at the time of synthesis of potent
DNA-alkylating metabolites by the host. It repairs double-stranded DNA at O6-
alkylguanine (Fig. 7.3a). MtOGT protects the M.tb DNA from GC to AT transition
mutations associated with O6-alkylated guanine in DNA (Miggiano et al. 2013).
Gene encoded by MtOGT is part of the operon involved in adaptive response.
Fig. 7.3 (a) Double-stranded DNA repair by MtOGT (O6-methylguanine MTase; Rv1316c),
(b) epigenetic modulation by M.tb MTases (MamA, MamB and HsdM)
Structural features of MtOGT were shown by Miggiano et al. (2013) where they
reported the structural conserved nature of this enzyme. Point mutations (T15S and
R37L) in MtOGT were observed in geographically distributed isolates of M.tb and
various drug-resistant strains. These mutational variations could be correlated with
defect in alkylated DNA repair and equilibrium between genome stability and
adaptability to the host in due course of evolution. In vitro activity analysis of
MtOGT and the two-point mutations associated with it showed impaired activity
of the mutated proteins in terms of alkylated DNA damage repair. MtOGT R37L
mutant exhibited tenfold lesser affinity for methylated double-stranded DNA as
compared to wild-type protein. Further crystal structure analysis of MtOGT and
MtOGT-R37L showed huge structural conservation of members of this protein
family (Miggiano et al. 2013).
In 2016, Miggiano and colleagues further revealed a peculiar structural assembly
of this MtOGT on the monoalkylated dsDNA molecule, shedding more light towards
the mechanisms of protein clustering at sites of damaged DNA and disassemble of
protein-DNA complexes at the time of repair (Miggiano et al. 2016).
7.2.2.2 DNA Methylation Leading to Epigenetic Regulation

DNA methylation performs regulatory function in a number of prokaryotic
pathogens but has not been broadly deciphered in mycobacteria. The three predicted
DNA MTases encoded by the genome of M.tb are MamA (Rv3263), MamB and
HsdM (Rv2756c) (Fig. 7.3b). These are responsible for the methylation of the
adenine at sixth position, out of which MamA has been very well characterized.
Interestingly none of these are linked with an equivalent restriction endonuclease.
A recent study, investigated the pattern of methylation in 12 different isolates of the
M.tb complex using PacBio technology and described the presence of three unique
methylation motifs which are associated with their respective MTases (Zhu et al.
2016). The study also identified a number of isolates where complete absence of
methylation of specific motifs in the genome was observed. It was proposed that loss
of function mutations in MTases led to the removal of methylation from the genome.
Another recent study, highlighted the lineage-specific methylome of M.tb which is
globally associated with kind of mutations present in the MTase genes (Phelan et al.
2018).
A comprehensive study by Shell et al. (2013) described one of the DNA MTases,
Rv3263 gene encoding active DNA adenine MTase MamA (mycobacterial adenine
methyltransferase). They reported that MamA methylates DNA at a six base-pair
sequence (CTGGAG) in a strain-specific manner in M.tb genome affecting expres-
sion of several genes. The genes affected by MamA shared overlapping methylation
site and sigma factor binding site as shown by mapping the transcriptional start site.
This report highlighted that MamA deletion resulted in reduction of survival of M.tb
in hypoxia and also that MamA is one of the predominant DNA MTases present in
M.tb strain H37Rv. These findings proposed MamA as an important mediator of
adaptation to different physiological stress (Shell et al. 2013).
Rv3204, one of another possible DNA MTase, encodes 101aa long protein and
shows close homology with other bacterial MTases (Mycobrowser) (Kapopoulou
et al. 2011). Rv3204 is also present in other Mycobacterium lineages like M. bovis,
M. leprae, M. marinum and M. smegmatis.
Overall, DNA MTases in M.tb have been shown to play a significant role in
tricking the host and ensuring its survival even in adverse conditions.
7.2.3 RNA Methyltransferases
When it comes to RNA MTases in M.tb genome, a total of 18 are present in M.tb
genome (Grover et al. 2016). RNA MTases are responsible for methylation of
nucleotides of mRNA, rRNA and t-RNA at specific positions and affect the RNA
metabolism in several ways such as pre-mRNA processing, post-transcriptional
maturation, translation initiation and resistance to antibiotics (Shatkin and Manley
2000; Bussiere et al. 1998).
Modification of rRNAs after transcription is highly important and conserved
phenomena and present in almost every kingdoms of life. In prokaryotes, rRNAs
are usually modified by methylation by two different ways: (i) methylation of the
hydroxyl group of pentose sugar and (ii) methylation of purine or pyrimidine bases.
These kinds of methylation modifications increase the chemical and functional
interaction of ribosomes and provide structural integrity to ribosomes by increasing
RNA-protein interactions of 30S and 50S subunit assembly.
7.2.3.1 Functions of Different RNA MTases on the Basis of Structural

Analysis
X-ray crystallographic structures of a number of SAM-MTases modifying protein,
DNA, RNA, and small molecules have been reported. Regardless of very less
sequence homology amongst several families of SAM-dependent MTases, majority
of them have a structurally conserved catalytic SAM-binding motif.
One such SAM-dependent MTase of M.tb, RNA methyltransferase (Rv2118c),
has signature sequence motifs which are conserved throughout the SAM-MTase
family. Its crystal structure revealed a homotetrameric structure with the presence of
N-terminal domain and C-terminal domain. The larger domain, C-terminal, binds the
cofactor AdoMet, and the N-terminal domain mainly contains β-structure with an
additional fold, absent in other MTases of known structures (Gupta et al. 2001). In
this report, Rv2118c was suggested to be an RNA methyltransferase on the basis of
structure analysis and homology to yeast Gcd14p. Later, Varshney et al. confirmed
the function of Rv2118c as a t-RNA MTase forming N1-methyladenosine of a highly
conserved adenine at position 58 in the TΨC loop of t-RNA which helps in stabili-
zation of t-RNA (Varshney et al. 2004). This kind of base modification of t-RNA
acts as a virulence factor and growth determinant in pathogenic bacteria.
Rv2966c another RNA MTases of M.tb was identified as ortholog of ribosomal
RNA small subunit methyltransferase D (RsmD) protein of E. coli. They showed
that Rv2966c can complement RsmD-deleted E. coli cells, and its crystal structure
revealed structural homology to RsmD. Recombinant Rv2966c uses 30S ribosomes
as substrate and methylates guanidine of 16S rRNA at 966 position. Crystal structure
of the protein suggested a two-domain structure with a short hairpin domain at the
N-terminal and a SAM-MTase containing C-terminal domain. N-terminal domain is
involved in target recognition and plays an important role in ensuring specific
interactions with RNA near the P-site of the ribosome. The study further revealed
that this domain is also able to bind to DNA; this property may perhaps be used
under particular cellular growth stages (Kumar et al. 2011). However, latest report
by Sharma et al. contradicts the function of Rv2966 by proving it as a DNA MTase.
According to their study, Rv2966 is a 5-methylcytosine-specific DNA MTase, which
secretes out from the M.tb after infection and localizes to the nucleus of the host cell.
This MTase epigenetically modulates the host machinery by interacting not only
with the host chromatin but also with the histones H3 and H4. DNA methylation
activity of Rv2966 is predominantly in non-CpG context and dependent on its
phosphorylation by mycobacterial kinases (Sharma et al. 2015).
Rv2372c is an RsmE-like MTase of M.tb which methylates N3 position of uridine
1498 in 16S rRNA. Methylated U1498 exists in decoding centre of ribosomes. A
point mutation in U1498 inhibits formation of the first peptide bond at the time of
translation. The crystal structure of Rv2372c revealed the presence of core fold of
SPOUT superfamily MTases which is active in its dimeric form. The study proved
that Rv2372c is orthologous to RsmE which is able to complement RsmE-deleted
E. coli cells. A ternary model of Rv2372c with 16S rRNA fragment and cofactor
SAM identified an RNA binding site at RNA interphase and SAM-binding pocket in
the deep trefoil knot. The involvement of U1498 in translation fidelity as well as
hygromycin resistance provides greater importance of its methylation (Kumar et al.

2014).
7.2.3.2 RNA MTases in Drug Resistance

Most of the antibiotics target ribosomes in the cell, and mutations at these sites result
in antibiotic resistance. One such notorious candidate erm (Rv1988), probable rRNA
MTase, has been shown to confer macrolide antibiotic resistance (Buriankova et al.
2004). In this report, through in silico studies, Rv1988 was suggested to be a
macrolide-lincosamide-streptogramin (MLS) resistance gene, from the erythromycin
resistance rRNA methylase (erm) family encoding 23S rRNA MTase. Further
thorough experimental studies proved the gene to be conserved in the M.tb complex.
Additionally, the gene was reported to show a high level of resistance through target
modification.
Another example of involvement of RNA MTases in drug resistance is mutations
in tlyA (Rv1694) gene encoding an O-methyltransferase which confers capreomycin
resistance. Capreomycin is a cyclic aminoglycoside-like peptide antibiotic which
binds the ribosome at the interface of large and small subunits (Johansen et al. 2006).
Proper methylation by TlyA is prerequisite for the optimum binding of this drug and
consequent inhibition of ribosomal activity. TlyA methylates ribose 2-hydroxy
group of two cytidine residues, C1409 of 16S rRNA, present inside the 30S subunit
of ribosome also known as decoding centre, and C1920 of 23S rRNA, which is
found in a highly conserved region of the 50S subunit of ribosome close to the
interface of both the subunits. This dual substrate activity (16S and 23S rRNA) of
TlyA is conducted by two stable structural domains connected by a linker region,
where N-terminal domain binds with the rRNA and C-terminal domain binds with
the SAM, a methyl donor. By X-ray crystallographic studies, it was revealed that this
inter-domain linker provides a structural plasticity which imparts TlyA to recognize
its two structurally distinct rRNA substrates (Witek et al. 2017).
Reduced streptomycin resistance in clinical isolates of M.tb is associated with
mutations found in gidB gene (Rv3919c) which is also an rRNA MTase. GidB
methylates the N7 atom of guanine 518 in 16S rRNA. G518 methylation structurally
stabilizes the 16S rRNA during protein translation by interacting with S12 protein of
ribosome. Proline 44 of S12 interacts with mRNA at third wobble position making
direct contact to the first two nucleotide of the codon anticodon helix (Ogle et al.
2001). Streptomycin, an aminoglycoside antibiotic, binds to a region in small
ribosomal subunit known as accuracy centre and inhibits translation of proteins.
Deletion of GidB gene from pathogenic strain of M.tb showed alteration in methyla-
tion of rRNA and subsequently interrupted the binding of streptomycin to its 16S
rRNA substrate eventually generating the streptomycin resistance phenotype (Wong
et al. 2013).
7.2.3.3 Dual Nature RNA MTases

The presence of multiple functional domains can lead to dual nature of proteins and
can switch or multitask when required in specific environments. This interesting
mechanism exists in M.tb MTases as well.
Rv1694 of M.tb also denoted as tlyA when expressed in E.coli showed haemolytic
activity, processes of rupturing red blood cells (Wren et al. 1998). The product of this
TlyA was annotated as a haemolysin. They also reported the presence of TlyA
homologs in Mycobacterium avium, Mycobacterium leprae and Mycobacterium
bovis BCG but lacking in non-pathogenic strain M. smegmatis. TlyA name comes
from the spirochaete Serpulina hyodysenteriae, where it acts as main factor in swine
dysentery pathogenesis (Hyatt et al. 1994).
Later Rv1694 was showed to function as ribosomal RNA MTase by Johansen
et al. (2006). They showed this tlyA encoding 20 -O-methyltransferase that resulted in
modification of C1409 nucleotide in helix 44 of 16S rRNA and C1920 nucleotide in
helix 69 of 23S rRNA. This resulted in conferring resistance against the antibiotics
capreomycin and viomycin.
In order to shed more light about dual nature of Rv1694, Rahman et al. reported
the diverse functions of this MTase that included haemolytic activity and ribosomal
RNA methylation. The study claimed Rv1694 is present inside the cell to methylate
the ribosomal RNA and on the cell membrane to destabilize the target membranes
(Rahman et al. 2010).
In general, the activity of MTases in M.tb has not been widely studied, but the
data available in the literature points us to the potential roles of DNA and RNA
MTases in enabling the M.tb survival in the range of extreme environment and
helping the bacterium to develop multidrug resistance and maintain the diversity in
strain-dependent manner.
7.2.4 Protein Methyltransferase
Methylation of protein is a kind of post-translational modification where addition of

methyl group on the arginine or lysine amino acid residues of a protein sequence
occurs. Protein methylation of histone proteins has been widely studied where
addition of methyl group can epigenetically activate or repress the expression of
certain genes.
Heparin-binding haemagglutinin (HBHA) and laminin-binding protein (LBP) are
methylated surface-localized adhesins that facilitate the interaction of the M.tb with
the non-phagocytic cells. C-terminal of HBHA, the active domain of the protein,
shows a complex methylation pattern in the form of mono- and dimethyl lysines
which protects it from proteolysis and affects the biochemical and immunological
properties of this protein. Methylation of HBHA was absent when recombinant
protein was expressed in E.coli cells (Pethe et al. 2000).
In a recent report, M.tb MTase Rv1988 has been found to be epigenetically
modulating the host machinery by histone methylation. Rv1988 is part of the M.tb
secretome and present only in pathogenic species of genus Mycobacterium. This
MTase localizes to the nucleus of the host after infection and methylates histone
H3 at arginine 42 position and subsequently downregulates the expression of the
host genes involved in generation of immune response against pathogen. Deletion
mutant of Rv1988 showed reduced survival inside the host proving it as an important
virulence factor of M.tb which exploits the host epigenetic machinery in a nonca-
nonical manner (Yaseen et al. 2015).
7.2.5 Methyltransferases Involved in Phenolglycolipid

Biosynthesis
Phthiocerol dimycocerosates (DIM), glycosylated phenolphthiocerol and acid

methanolysates are the three characteristic waxes of M.tb. Dimycocerosates of the
phthiocerol detected in the free lipids along with glycosylated phenolphthiocerol,
also known as phenolglycolipid (PGL), are two important groups of virulence
factors in mycobacterial species, which are involved in pathogenesis in humans.
Phthiotriol/phenolphthiotriol dimycocerosates are encoded by the gene Rv2952.
Through insertional mutation analysis, it was found that the gene product is an
MTase that catalyses the methyl groups’ transfer onto the intermediate compounds
such as phthiocerol and glycosylated phenolphthiotriol dimycocerosate to form
phthiocerol dimycocerosates (DIM) and glycosylated phenolphthiocerol
dimycocerosates (PGL) and other related p-hydroxybenzoic acid derivatives
(p-HBAD). The gene has the similar sequence motif as the MTase which methylates
the hydroxyl groups of fatty acids.
Phthiocerol dimycocerosates of M.tb play a major role in virulence primarily in
the initial stages of infection when the mycobacterium encounters the host
macrophages. Mutation of genes in the DIM biosynthesis pathway proved that
DIM is involved in the receptor-dependent phagocytosis of M.tb and also inhibits
the phagosomal acidification. DIM regulates M.tb invasion into host macrophages
by altering organization of the lipid of the macrophage membrane, thus altering the
biophysical properties of the membrane. The DIM induces changes in the lipid order
and alters the efficiency of receptor-mediated phagocytosis by M.tb. These changes
in lipid control the phagosomal pH further placing bacilli in a protecting
environment.
The enzymes encoded by genes Rv2955c, Rv2954c and Rv2956 are involved in
the methylation process of phenolic glycolipids, produced by M.tb. They are neces-
sary for O-methylation of the fucosyl residue of PGL-tb. These molecules impart
pathogenicity by regulating the response of the immune system of the host during
infection. The genes Rv2954c, Rv2955c and Rv2956 were found to be MTases by the
expression and purification analysis of phenolglycolipids produced by mutants of M.
tb harbouring a deletion of these genes. It was revealed that the hydroxyl groups
located at the positions 2, 3 and 4 of the fucosyl residue of PGL-tb were
O-methylated. It was also established that the methylation process occurs in a
sequential manner starting from the position 2, followed by 3 and 4 (Simeone
et al. 2013).
An important variant of phenolic glycolipids in M.tb is PGL-tb which consists of
an enormous-sized lipid core wrapped up by a glycosylated aromatic nucleus. The
carbohydrate region playing a significant role in pathogenicity comprises three
residues of sugar and two rhamnosyl units with a terminal fucosyl residue, which is
per-O-methylated. This methylation imparts a significant pathogenic characteristic
to the glycolipid component.
7.3 Conclusion
The huge number of MTases in M.tb has gained widespread attention from the TB
researchers due to their significant roles in pathogenesis, virulence and adaptation
inside the extreme environment of the host (Fig. 7.4). Interestingly, methylation of
biomolecules is now acknowledged as a dynamic modification enhancing the com-
plexity of interactions amongst DNA, RNA, protein and other components of
cellular machinery. Methylation all together regulates biomolecular interactions,
surface properties of the cell, structure and functional dynamics of chromatin and
expression of genes. It has been estimated that 1% of total genes code for MTases in
human genome where it is 3% in the case of M.tb, i.e. 121 in total number, and
majority of them are uncharacterized so far. It is therefore important to understand
the role of many of the uncharacterized MTases in pathomechanism of TB. In
addition, understanding the potential of these MTases as drug targets would enable
designing more effective treatment strategies to combat TB. New development on
TB treatment should take into consideration the molecules that can uniquely inhibit
M.tb MTases.
Fig. 7.4 Functional insights into the Mycobacterial methyltransferases and their site of action
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The PE and PPE Family Proteins
of Mycobacterium tuberculosis: What they 8
Are Up To?
Ravi Pal, Faiza Nazar, and Sangita Mukhopadhyay
Abstract
An ebb and flow journey of over 125 years since the discovery of Mycobacterium
tuberculosis (M.tb), the causative agent of tuberculosis (TB), clearly established it
as an extremely resilient and elusive pathogen. Accurate diagnosis and effective
treatment and full control of this dreaded pathogen are still beyond our reach due
to its complex pathobiology. Other features like latency, emergence of multiple
drug-resistant strains, and co-occurrence of TB with HIV further complicate
development of effective vaccines and drugs to treat TB. Complete genome
sequence of M.tb has identified a unique set of proteins belonging to PE and
PPE family, which is characterized by the presence of highly conserved proline–
glutamate (PE) and proline–proline–glutamate (PPE) residues at the N-terminal
conserved domain. Ever since its discovery, the PE/PPE family has been the
focus of attention in the field of TB research. The PE and PPE family of proteins
appears to be critical for mycobacterial survival which modulates the protective
innate and adaptive immune responses of the host. In this chapter, we summarize
the findings on evolution, characterization, localization, and transcriptional regu-
lation of the PE and PPE genes. We also describe the role of PE and PPE proteins
in the modulation of host immune responses. A better understanding of the PE
and PPE proteins may lead to the development of innovative and improved anti-
TB therapeutics, vaccines, and diagnostics in the future.
R. Pal
Laboratory of Molecular Cell Biology, Centre for DNA Fingerprinting and Diagnostics (CDFD),
Hyderabad, Telangana, India
Manipal Academy of Higher Education, Manipal, Karnataka, India
F. Nazar · S. Mukhopadhyay (*)
Laboratory of Molecular Cell Biology, Centre for DNA Fingerprinting and Diagnostics (CDFD),
Hyderabad, Telangana, India
e-mail: sangita@cdfd.org.in

https://doi.org/10.1007/978-981-32-9413-4_8
124 R. Pal et al.
Keywords
Mycobacterial PE and PPE family of proteins · Evolution · Innate and adaptive
immune responses · Vaccines · Virulence
Abbreviations
BCG bacillus Calmette–Guérin

M.tb Mycobacterium tuberculosis
PE proline–glutamate
PPE proline–proline–glutamate
TB tuberculosis
TLR Toll-like receptor
8.1 Introduction
Mycobacterium tuberculosis (M.tb) discovered by Robert Koch in 1882 is an

obligate pathogenic bacterium. It is a part of family Mycobacteriaceae. Measured
growth rate, hardy cell wall, and various other sneaky strategies adopted by the
bacilli not only help it to survive inside the host but also to modulate the host
immune response, thus creating a promising environment for further infection.
Regardless of bacillus Calmette–Guérin (BCG) vaccination and directly observed
treatment, short course (DOTS) therapy, tuberculosis still persists in the population
of developing countries (WHO Global tuberculosis report 2017). Additionally,
appearance of multidrug-resistant (MDR) and extensively drug-resistant (XDR)
strains has intensified the illness caused by the bacillus. It causes deaths among
millions of people every year worldwide. According to annual WHO report (2017),
10.4 million new TB cases were estimated, out of which 6.2 million were men,
3.2 million were women, and 1.0 million were children. In 2016, there were
1.7 million deaths due to TB (1.3 million HIV-negative people and 0.4 million
HIV-positive people) (WHO Global tuberculosis report 2017). There is an indication
that mortality of TB has fallen down to 37% since 2000.
With the annotation of complete M.tb H37Rv genome, around 4000 genes (with
high GC content) were identified which led to the discovery of novel genes and gene
families (Cole et al. 1998). PE/PPE family of genes constitutes approximately 10%
of the M.tb genome with apparently no known biological functions. There are ~100
PE and ~70 PPE genes present in M.tb. Abundance of these genes has led to the
understanding of their role in the context of host–pathogen interaction. PE and PPE
proteins are named after the highly conserved proline–glutamate (PE) and proline–
proline–glutamate (PPE) residues, respectively, near the start site of their translated
proteins (Cole 1999). These genes code almost conserved N-terminal sequence of
110 and 180 amino acids in PE and PPE families (Fig. 8.1), respectively, (Cole
8 PE/PPE Family of Proteins of M. tuberculosis 125
Fig. 8.1 Diagrammatic representation of PE and PPE gene families. PE and PPE family proteins
possess a relatively conserved N-terminal of ~110 and ~180 amino acids (aa), respectively. These
proteins are further divided into subgroups based on the variable C-terminal
et al. 1998) while the variable C-terminal is believed to be a cause of antigenic and
genetic variations across the species. On the basis of variation in the length and
sequence, these proteins are further classified into subgroups. PE family is
subdivided mainly into two subgroups, i.e., PE_PGRS (polymorphic GC-rich
sequences) and PE (with no such distinctive feature). The PPE family encompasses
PPE_MPTR (major polymorphic tandem repeats), PPE_SVP (Gxx-SVPxxW motif),
and PPE_PPW (PxxPxxW motif) subgroups (Fig. 8.1). Probably, diversity within
these subgroups provides them the freedom to have different functions (Fishbein
et al. 2015).
In the present manuscript, we have briefly summarized evolution, variation, and
phylogeny of PE/PPE family of proteins and their roles in modulation of host
immune responses and M.tb pathogenesis. Additionally, their potential roles as
vaccines and use as diagnostic antigens in TB have been summed up.
8.2 Evolution of PE and PPE Genes
The PE/PPE family consists of around 100 and 70 members, respectively. The group
can further be divided into subgroups comprising of members whose length and
sequence features are varied. Multiple tandem repeats of Gly-Gly-Ala (or variant)
are characteristics of PE_PGRS (polymorphic GC-rich sequence), while the
polypeptides rich in repeats of Asn-X-Gly-X-Gly-Asn-X-Gly are present in
126 R. Pal et al.
PPE_MPTR subgroup (Gey van Pittius et al. 2006). PE/PPE gene clusters are
arranged in operons in M.tb genome. A total of 28 PE/PPE operons have been
identified which are catalogued in a way that most PE members are placed upstream
of PPE members. Only a few members of PE protein family contain relatively
smaller N-terminal domain, while most of them have C-terminal regions which
vary in size from 100 to 1400 residues (Cole et al. 1998).
PE/PPE family of genes is exclusively present in mycobacterial species and is
absent from the closest relative bacteria such as Nocardia farcinica (Ishikawa et al.
2004). Interestingly, pe and ppe genes are present predominantly in the pathogenic
species like M. marinum, M. avium, and M.tb but not in the nonpathogenic species
like M. smegmatis (Abdallah et al. 2009), indicating toward a biased evolutionary
selection of this family in pathogenic species mainly. Origin of PE/PPE family of
genes is not clearly understood, but there are speculations that earliest form of pe and
ppe genes was in close association with the ESX or type VII secretion system
(Abdallah et al. 2007). In M.tb, there are five esx gene clusters (Gey Van Pittius
et al. 2001), and phylogenetic studies indicate that esx gene clusters are expanded
due to the multiple gene duplication. Therefore, it is possible that duplication of esx
gene cluster has enabled duplication of closely associated pe and ppe genes
(Fig. 8.2). Presence of frequent homologous regions among pe and ppe genes
indicates that spontaneous homologous recombination might have played a crucial
role in generating intragenic variations among them (Gey van Pittius et al. 2006;
McEvoy et al. 2009, 2012). Insertions/deletions resulting in frame shift and
transposon-mediated insertions have certain roles in creating genetic variation.
Based on the phylogenetic study by Gey van Pittius et al. (2006), pe and ppe
genes were further classified into sub-lineages, and it was observed that certain
sub-lineages have more variations as compared to others. But the reason behind such
biased selection is still unknown. In silico studies too have revealed that pe and ppe
genes are more prone to mutations as compared to non-pe and non-ppe genes
(McEvoy et al. 2012). There are reports stating that insertion elements (IS elements)
have contributed to rearrangement of ppe genes (Sampson et al. 1999). IS6100, an
insertion element, has been found to bring genetic changes by inserting into pe and
ppe genes (Pérez-Lago et al. 2011).
8.3 Characterization, Localization, and Functional Studies

of PE and PPE Proteins
Identification and characterization of PE and PPE proteins are important to under-

stand their roles in M.tb metabolism and its pathophysiology. In fact, various
studies have indicated that the PE and PPE proteins regulate growth and survival
of pathogenic mycobacteria and modulate important host immune responses as
well. PE11 protein contains GXSG motif and shows esterase activity (Singh et al.
2016). PE11, an essential protein of M.tb, is involved not only in regulating the cell
wall architecture of mycobacteria but also in favoring mycobacterial replication
in vivo (Singh et al. 2016; Rastogi et al. 2017). In silico studies by Sultana et al.
Fig. 8.2 Flow diagram predicting evolution and expansion of pe and ppe genes. During the course
of evolution, pe and ppe genes were first inserted into the esx gene cluster. Duplication of esx cluster
led to the duplication of pe and ppe genes. Spontaneous homologous recombination, random
mutation, insertion/deletion, and IS elements contributed to the expansion and evolution of pe
and ppe genes into different subgroups
(2016) reveal that certain PE and PPE proteins like PE1, PE2, PE3, PE4, PE16,
PPE28, PPE42, and PPE63 have hydrolase activity domain. PE and PPE proteins
have been detected in cell filtrate, mycobacterial membrane, and/or cell wall
fraction. N- and C-terminals of the protein play an important role in the localization
of the protein. In Mycobacterium (Cascioferro et al. 2007; Zumbo et al. 2013), the
N-terminal PE domain of the well-defined PE_PGRS33 was found to be important
for protein localization to the cell wall. Cascioferro et al. (2011) confirmed that first
30 amino acids of PE domain in PE_PGRS33 are crucial for its surface localiza-
tion. Interestingly, in Mycobacterium marinum, processed PE domain of
PE_PGRS33 was secreted into the culture medium when expressed alone. Chatrath
et al. (2014) have demonstrated that the PE_PGRS domain of PE_PGRS30 is
responsible for its localization at bacterial cell poles. Association of several PE and
128 R. Pal et al.
PPE proteins with the mycobacterial cell wall suggests their involvement in
structural integrity and colony morphology of the bacilli. For example, when
PE_PGRS30 was expressed in nonpathogenic fast-growing M. smegmatis, colony
size decreased (Chatrath et al. 2011). Another protein, PE11, when expressed in
M. smegmatis, exhibited altered colony morphology and caused changes in the
lipid profile of cell envelope with higher amounts of glycolipids and polar lipids
(Singh et al. 2016). Structural integrity of the cell wall was also compromised in
ppe38-deleted M. marinum due to alteration of cell surface, and as a result, biofilm
formation was interrupted (Dong et al. 2012). Many PPE genes have roles in
maintaining the cell wall integrity as was shown by disrupting some of the ppe
genes by transposon mutagenesis that leads to increased resistance against ampi-
cillin (Danilchanka et al. 2008). Ramakrishnan et al. (2016) observed that if the
ppe27-pe19 locus is deleted, the sensitivity of M.tb cell wall to stress increases
suggesting their roles in strengthening the bacterial cell wall to withstand stress
conditions in the host. An interesting study by Ates et al. (2018) reveals that
mutation in the PPE38 protein completely blocked secretion of more than
80 proteins of PPE_MPTR and PE_PGRS subsets and made the mutant more
virulent than the wild type. Importantly, hypervirulent clinical M.tb strains of the
Beijing lineage have such a mutation.
Direct interaction of PE and PPE proteins with host depends on their surface
exposure or secretion into the extracellular milieu. Some of the proteins of PE and
PPE family are cell wall localized and therefore can directly interact with host cell
surface molecules. PPE18 and PPE17 are present on M.tb cell wall and interact with
Toll-like receptor 2 (TLR2) (Nair et al. 2009; Udgata et al. 2016). While PPE17
interacts with the leucine-rich repeat (LRR) 16–20 domain of TLR2 inducing a
pro-inflammatory response, PPE18 very strongly binds to LRR 11–15 domain of
TLR2 and activates mainly the non-protective anti-inflammatory response. Detec-
tion of PPE41 in late endosomes (Abdallah et al. 2006) and PPE2 in macrophage
nucleus (Bhat et al. 2017) suggests that some family members are secretory in
nature. High-throughput proteomics data have also confirmed the localization of
PE and PPE proteins on the M.tb cell wall and beyond. A detail knowledge of
cellular localization and spatial and temporal regulation of PE/PPE proteins is likely
to provide important clues on host–pathogen interaction and designing of novel anti-
TB therapeutics.
The localization and functions of important PE and PPE proteins are listed in
Table 8.1.
Table 8.1 Localization and functions of some PE and PPE proteins

Name Location Function
PE2 Membrane (Mawuenyega et al. 2005) Belongs to the esterase family;
(Rv0152c) hydrolyzes short-to-medium-chain
p-nitrophenyl esters (Bala et al. 2018)
PE3 Membrane (Målen et al. 2011) PE3 stimulates TNF-α, IL-6, and IL-2
(Rv0159c) cytokine and provides protection
against M.tb in mice (Singh et al.
2013)
PE5 PE5 – culture filtrate (Målen et al. Upregulates IL-10 and downregulates
(Rv0285) 2007) pro-inflammatory responses; supports
enhanced bacterial survival in
macrophages (Tiwari et al. 2012)
PE6 Cell wall (Mawuenyega et al. 2005) Essential gene. Function unknown
(Rv0335c)
PE9 Membrane (Mawuenyega et al. 2005) PE9 (Rv1088)–PE10 (Rv1089)
(Rv1088) protein pair forms heterodimers;
induces macrophage apoptosis
through TLR4 (Tiwari et al. 2015)
PE11 (lipX) Membrane (Målen et al. 2011; de Cell wall remodeling and
(Rv1169) Souza et al. 2011) mycobacterial virulence (Singh et al.
2016)
PE12 Membrane (Målen et al. 2011) Induces high percentage of CD8+ T
(Rv1172c) cells secreting IFN-γ and TNF-α
(Stylianou et al. 2018)
PE13 Cell wall (Li et al. 2016) Enhanced survival in THP-1
(Rv1195) macrophages; upregulates IL-6 and
IL-1β; downregulates SOCS3;
modulates p38-ERK-NF-κB
signaling; induces apoptosis in THP-1
cells (Li et al. 2016)
PE14 Cell wall (Wolfe et al. 2010) Nonessential gene. Function unknown
(Rv1214c)
PE15 Membrane (Målen et al. 2011; de Enhances survival of Mycobacterium
(Rv1386) Souza et al. 2011) in macrophages (Tiwari et al. 2012)
PE17 Whole cell lysate (de Souza et al. Nonessential gene. Function unknown
(Rv1646) 2011)
PE20 Membrane (de Souza et al. 2011) Nonessential gene. Function unknown
(Rv1806)
PE25 Cell wall (Wolfe et al. 2010), PE25/PPE41 dimer induces necrosis
(Rv2431c) membrane (Målen et al. 2011; de in murine macrophages (Tundup et al.
Souza et al. 2011) 2014), helps in activation and
maturation of DCs; favors Th2
response; and induces strong humoral
response in mice (Chen et al. 2016)
PE27 Membrane (Målen et al. 2011) Induces DC maturation by
(Rv3018A) up-regulating CD80, CD86, MHC
class I, and MHC class II; upregulates
TNF-α, IL-1β, IL-6, and IL-12p70.
Induces IFN-γ-producing memory T
cell responses in M.tb infected mice;
contributes to Th1-polarization
(Kim et al. 2016)
(continued)
130 R. Pal et al.
Table 8.1 (continued)

PE31 Membrane; lysate not culture filtrate Nonessential gene. Function unknown
(Rv3477) (de Souza et al. 2011)
PE35 PE35 – culture filtrate (Målen et al. Co-operonic with PPE68; upregulates
(Rv3872) 2007) IL-10 by interaction with TLR2 via
MAPK pathway; stimulates MCP-1 in
THP-1 macrophages (Tiwari et al.
2014)
PE_PGRS11 Cell wall (Chaturvedi et al. 2010) Interacts with TLR2 and imparts
(Rv0754) resistance to oxidative stress by
upregulating COX-2 and Bcl2.
Differential B cell responses during
TB infection in human sera
(Chaturvedi et al. 2010)
PE_PGRS12 Membrane (Mawuenyega et al. 2005) Nonessential gene. Function unknown
(Rv0832)
PE_PGRS29 Membrane (Målen et al. 2011; Nonessential gene. Function unknown
(Rv1468c) de Souza et al. 2011)
PE_PGRS30 Bacterial cell pole (De Maio et al. Colonizes lung tissue, causes tissue
(Rv1615c) 2014) damage; inhibits phagosome-
lysosome fusion in macrophage; and
induces cell death in mice (Iantomasi
et al. 2012)
PE_PGRS33 Cell wall (Minerva et al. 2017) Helps in entry into macrophages via
(Rv1818c) TLR2 interaction (Palucci et al. 2016);
induces TNF-α via TLR2 signaling
(Zumbo et al. 2013); activates humoral
response in mice and human (Cohen
et al. 2014)
PE_PGRS38 Membrane (Målen et al. 2011) Nonessential gene. Function unknown
(Rv2162c)
PE_PGRS39 Membrane (Mawuenyega et al. 2005) Nonessential gene. Function unknown
(Rv2340c)
PE_PGRS56 Membrane (Mawuenyega et al. 2005) Induces IFN-γ in (human peripheral
(Rv3512) blood mononuclear cells) PBMCs
(Nebenzahl-Guimaraes et al. 2017)
PE_PGRS62 Cell wall (Thi et al. 2013) Blocks phagosome maturation and
(Rv3812) inhibits iNOS expression (Thi et al.
2013); reduces phagolysosome
maturation (Huang et al. 2012)
PPE2 Culture Filtrate (Bhat et al. 2013) Downregulates NO production by
(Rv0256c) inhibiting inos gene transcription
(Bhat et al. 2017)
PPE4 Membrane (Mawuenyega et al. 2005) PPE4 is a part of the ESX-3 locus; role
(Rv0286) in iron acquisition and virulence
(Tufariello et al. 2016)
PPE6 Cytosol, cell wall, and membrane Nonessential gene. Function unknown
(Rv0305c) (Mawuenyega et al. 2005)
(continued)

PPE7 Membrane (Díaz et al. 2017) Role in cell invasion in humans (Díaz
(Rv0354c) et al. 2017)
PPE11 Cytosol (Mawuenyega KG et al. Promotes cell death in THP1
(Rv0453) Mawuenyega et al. 2005), Culture macrophages; upregulates TNF-α,
filtrate (Målen et al. 2007) IL-6, IL-1β and downregulates IL-10;
enhances bacterial survival in THP-1
and mice (Peng et al. 2018)
PPE17 Cell wall (Donà et al. 2013) Binds to TLR2 and induces TNF-α
(Rv1168c) (Udgata et al. 2016)
PPE18 Cell wall (Wolfe et al. 2010) Interacts with TLR2 and activates
(Rv1196) IL-10 cytokine (Nair et al. 2009).
Inhibits proinflammatory cytokines by
targeting the p38MAPK-SOCS3-Rel
signaling; skews the anti-PPD T-cell
response towards the Th2-type. (Nair
et al. 2011). Imparts better survival of
M.tb in mouse model of infection
(Bhat et al. 2012)
PPE19 Cell wall (Wolfe et al. 2010) Nonessential gene. Function unknown
(Rv1361c)
PPE20 Membrane (Målen et al. 2011) Nonessential gene. Function unknown
(Rv1387)
PPE25 Cell wall (Mi et al. 2017) Enhanced survival in murine
(Rv1787) macrophages and mice. Upregulates
TNF-α, IL-1β and downregulates
IL-6; activates NF-κB, p38MAPK,
and ERK signaling pathway (Mi et al.
2017)
PPE26 Membrane (Målen et al. 2011; Interacts with TLR2 and activates
(Rv1789) de Souza et al. 2011) MAPK and NF-κB signaling
pathways; stimulates production of
proinflammatory cytokines TNF-α,
IL-6, and IL-12p40 in murine
macrophages (Su et al. 2015)
PPE27 Cell wall (Yang et al. 2017) Exhibits higher survival rate under
(Rv1790) several hostile conditions in vitro;
longer persistence in mouse tissues.
Upregulates TNF-α, IL-1β, nitric
oxide (NO) and downregulates IL-6 in
mice macrophages (Yang et al. 2017)
PPE29 Cell wall (Wolfe et al. 2010) Nonessential gene. Function unknown
(Rv1801)
PPE31 Membrane (Målen et al. 2011; Confers the basal level of M.tb
(Rv1807) de Souza et al. 2011) resistance to vancomycin (Provvedi
et al. 2009)
(continued)
132 R. Pal et al.

PPE32 Membrane (Målen et al. 2011; Promotes proinflammatory cytokines
(Rv1808) de Souza et al. 2011) and host cell apoptosis (Deng et al.
2016)
PPE33 Membrane (Målen et al. 2011; de Nonessential gene. Function unknown
(Rv1809) Souza et al. 2011)
PPE34 Surface exposed (Sampson et al. Interacts with TLR2 and triggers
(Rv1917c) 2001) functional maturation of human DCs
(Bansal et al. 2010)
PPE36 Surface exposed (Mitra et al. 2017) Role in nutrient acquisition (Mitra
(Rv2108) et al. 2017)
PPE37 Cell wall (Ahmad et al. 2018) Downregulates TNF-α, IL-6,
(Rv2123) IL-12p70, and IL-1β. Lower levels of
NF-κB, ERK, and p38 MAPK in
murine macrophages (Daim et al.
2011)
PPE38 Membrane (Målen et al. 2011) Manipulates innate immune response
(Rv2352c) of host and is shown to play a role in
phagocytosis and mycobacterial
(M. marinum) virulence (Dong et al.
2012)
PPE39 Membrane (Målen et al. 2011) Protective role against Beijing/K
(Rv2353c) strain in mice (Kim et al. 2017)
PPE40 Whole cell lysate (de Souza et al. Essential gene. Function unknown
(Rv2356c) 2011)
PPE41 Culture filtrate (Målen et al. 2007) PE25/PPE41 dimer induces necrosis
(Rv2430c) in murine macrophages (Tundup et al.
2014); helps in activation and
maturation of DCs; favors Th2
response and strong humoral response
in mice (Chen et al. 2016)
PPE44 Cell wall (Yu et al. 2017) Induces apoptosis; upregulates IL-6
(Rv2770c) and IL-12p40 in THP-1 macrophages;
and enhances intracellular survival
(Yu et al. 2017)
PPE51 Cell wall (Wolfe et al. 2010); Function unknown
(Rv3136) membrane (Målen et al. 2011)
PPE57 Cell wall (Xu et al. 2015) Induces macrophage activation and
(Rv3425) drives Th1-type immune responses
through TLR2 in mice (Xu et al. 2015)
PPE58 Cell membrane (Mawuenyega et al. Function unknown
(Rv3426) 2005)
PPE60 Membrane (Målen et al. 2011) Drives Th1/Th17 responses via Toll-
(Rv3478) like receptor 2-dependent maturation
of dendritic cells in mice (Su et al.
2018)
PPE62 Surface exposed (Mitra et al. 2017) Role in nutrient acquisition (Mitra
(Rv3533c) et al. 2017)
PPE63 Membrane (Mawuenyega et al. 2005) Nonessential gene. Function unknown
(Rv3539)
(continued)

PPE65 Cell wall (Qureshi et al. 2019) Binds to TLR2 and upregulates TNF-α
(Rv3621c) and IL-6 in THP-1 macrophages
(Qureshi et al. 2019)
PPE68 Membrane (Målen et al. 2011; de Co-operonic with PE35 modulates
(Rv3873) Souza et al. 2011) MAPK signaling pathways;
upregulates IL-10 in dose-dependent
manner in THP-1 macrophages
(Tiwari et al. 2014)
8.4 Effects of PE and PPE Proteins on Host Immune Responses
PE and PPE proteins modulate both innate and adaptive immune responses of the
host (Fig. 8.3). Various immune effector functions that are modulated/manipulated
by PE/PPE family of proteins are described below.
8.4.1 Innate Immune Responses
Innate immunity is the first component of the immune system which counteracts
various microorganisms. Innate immune cells detect the presence of microorganism
through pathogen-associated molecular patterns (PAMPs) and trigger downstream
effector signaling cascades to eliminate the infection. These are followed by induc-
tion of another tier of the immune system, i.e., adaptive immune response.
M.tb infection is initiated by inhalation of aerosol droplets containing small
numbers of bacilli (Smith 2003). Through the respiratory passage, bacilli reach to
the lungs and are internalized by resident alveolar macrophages through phagocyto-
sis. Macrophages are one of the important cells of the innate immune response which
during M.tb infection encounter the bacilli and induce an array of antimicrobial
mechanisms like secretion of antibacterial pro-inflammatory cytokines (TNF-α,
IL-12, IL-1β), free radicals, nitric oxide (NO), and reactive oxygen intermediates
(ROI) and induction of apoptosis and lysosomal degradation (Van Crevel et al. 2002;
Hussain Bhat and Mukhopadhyay 2015). After being recognized by pattern recog-
nition receptors (PRRs), bacilli trigger both innate and adaptive immune response
(Qureshi and Medzhitov 2003). M.tb activates different families of pattern recogni-
tion receptors, majorly the TLRs and the nucleotide-binding oligomerization
domain-like receptors (NODs) (Ferwerda et al. 2005).
Initial reports depicting the role of PE and PPE genes in modulating innate
immune machinery come from M. marinum which has two PE_PGRS gene
homologues to Rv1651c and Rv3812 of M.tb. These proteins are involved in
granuloma formulation and enhanced survival of the bacilli inside macrophages
134 R. Pal et al.
Fig. 8.3 Diagrammatic representation of macrophage effector functions modulated by PE and PPE
proteins during Mycobacterium sp. infection. ROIs, reactive oxygen species; RNIs, reactive
nitrogen species; TLR, Toll-like receptor; MHC, major histocompatibility complex
(Ramakrishnan et al. 2000). There are many PE and PPE proteins which act as
virulence factors and affect various macrophage functions. For example, in BCG, a
pe_pgrs33 mutant has shown restricted entry into macrophages (Brennan et al. 2001)
suggesting its role in promoting uptake of bacilli and its colonization inside
macrophages during infection. Palucci et al. (2016) have shown that PE_PGRS33
contributes to M.tb entry in macrophages through TLR2-dependent manner. Similar
observations were made when PE_PGRS33 was expressed in nonpathogenic
M. smegmatis (Singh et al. 2008). An interesting study indicates that PE_PGRS30
is required for full virulence of M.tb as pe_pgrs30 mutant was impaired in its ability
to colonize lung tissue (Iantomasi et al. 2012). Camacho et al. (1999) have shown
that attenuated PPE46 resulted in reduced M.tb growth in vitro. In macrophages,
phagosome maturation and acidification is a crucial defense mechanism for killing of
intracellular pathogens, and PE/PPE proteins can influence this process (Jha et al.
2010). For example, four pe_pgrs mutants and three ppe_mptr of BCG were found
to be predominantly localized within acidified phagosomes which reflects their
putative roles in inhibition of phagosome–lysosome fusion during infection (Stewart
et al. 2005). There are several reports depicting role of PE/PPE proteins in cell
viability. PE_PGRS5 localizes to endoplasmic reticulum and induces stress-
mediated apoptosis through TL-4 (Grover et al. 2018).
PE_PGRS33 induces apoptosis in murine macrophages through
TNF-α-dependent manner (Basu et al. 2007). Further, Cadieux et al. (2011) reported
that the PGRS region of PE_PGRS33 is required for its localization into
mitochondria but PE domain was crucial for induction of cell death. In
M. smegmatis, PE9 and PE10 were found to form a heterodimer and induce
apoptosis in THP-1 cells through TLR4 binding (Tiwari et al. 2015). In TB infection,
necrotic cell death is known to be important for dissemination of bacilli. PE25/
PPE41, a co-operonic protein complex, was found to induce necrosis in murine
macrophages (Tundup et al. 2014). Inflammasome activation is one of the important
events of M.tb infection. Inflammasome activation upregulates pro-inflammatory
cytokines like IL-1β and IL-18 which leads to pyroptosis (Briken et al. 2013). PE8
and PPE15 are part of ESX-5a, and it was observed that M.tb lacking ESX-5a was
not as efficient as the wild-type bacilli in activating inflammasome but did not affect
host cell death (Shah et al. 2015).
There are various ways by which PE/PPE proteins modulate macrophage effector
functions; few of them are reviewed and discussed here in detail.
8.4.1.1 Reactive Oxygen Intermediates (ROIs) and Reactive Nitrogen

Intermediates (RNIs)
During M.tb infection, activated macrophages produce reactive oxygen
intermediates (ROIs) via NADPH oxidase (NOX2/gp91phox) and reactive nitrogen
intermediates (RNIs) through inducible nitric oxide synthase (iNOS) (Ehrt and
Schnappinger 2009). Upon infection, superoxide ions (O2) are generated, which
are acted upon by dismutase to produce H2O2 which finally generates toxic hydroxyl
radicals (OH.) against the bacilli (Bedard and Krause 2007). RNIs are products of
inducible nitric oxide synthase (iNOS), an enzyme that synthesizes nitric oxide
(NO) from L-arginine in response to endogenous cytokines like IFN-γ and TNF-α
or pathogen-derived molecules. NO and other chemical intermediates, i.e., NO¯,
NO2¯, •NO2, N2O3, and N2O4, are involved in imparting resistance during myco-
bacterial infection. In addition to activated macrophages, polymorpholeukocytes
also generate ROIs and RNIs in response to M.tb infection (Hussain Bhat and
Mukhopadhyay 2015).
Mycobacterium has evolved to surpass these effector responses by having scav-
enger compounds like phenolic glycolipid I (PGL-1) in the thick cell wall (Launois
et al. 1989), enzyme catalase–peroxidase (KatG) neutralizing ROIs (Manca et al.
1999; Ng et al. 2004), superoxide dismutases SodA and SodC for neutralization
of.O2 (Piddington et al. 2001), and the peroxidase and peroxynitrite reductase
(Voskuil et al. 2011). An interesting study indicates that the PPE2 protein of M.tb
can suppress NO production in murine macrophages by downregulating expression
of inos gene resulting in increased survival inside macrophages (Bhat et al. 2013,
2017). PPE2 contains nuclear localization signal (NLS), which enables it to gain
entry into the nucleus and a DNA-binding motif which facilitates the DNA binding
at the promoter region of inos gene leading to its reduced transcription (Bhat et al.
2017). Furthermore, PPE2 reduces ROS levels in murine macrophages during
infection (Srivastava et al. 2019). PE_PGRS62, a protein expected to alter the cell
wall-related component, when expressed in M. smegmatis, has curtailed inos expres-
sion in infected macrophages without changing the transcriptional level by some
136 R. Pal et al.
unknown mechanism (Thi et al. 2013). PE11 (LipX or Rv1169c), which is

upregulated during oxidative stress in macrophages (Schnappinger et al. 2003) as
well as in patients with active TB (Rachman et al. 2006), modulates cell wall
architecture of the bacilli to resist free radicals and various environmental and
antibiotic stresses which is clearly reflected by the enhanced survival of
M. smegmatis expressing PE11 in murine macrophages as well as in mouse model
of infection (Singh et al. 2016). Other proteins, like PE5 and PPE15, suppress nitric
oxide production by downregulating inos at the transcriptional level and provide
protection to the pathogen against oxidative stresses (Tiwari et al. 2012).
8.4.1.2 Cytokine Signaling

Evidences from both experimental animal models and observation on patients have
firmly established the role of cytokines and their signaling pathways in mycobacterial
infection (Cooper and Khader 2008). During M.tb infection, macrophages secrete
cytokines like TNF-α, IFN-γ, IL-10, and IL-12 which play an important role in the
regulation of macrophage as well as other immune cell functions (Domingo-Gonzalez
et al. 2016). Cytokines have an important role in dictating the T cell response
phenotype. TNF-α, IL-12, and other pro-inflammatory cytokines enhance the
antibacterial defense and activate induction of the protective T helper (Th) 1-type T
cell response, whereas IL-10, the anti-inflammatory cytokine, is known to induce
Th2-type T cell response which causes decreased protection of the host and enhanced
mycobacterial survival. M.tb PPE17, a cell wall-localized protein, triggers a pro-
inflammatory-type response in macrophages by TLR1/2 heterodimerization through
PKCepsilon–IRAK3–p38MAPK signaling axis (Udgata et al. 2016), whereas PPE18,
another cell wall-localized protein, binds to TLR2 but to a different domain (LRR
11–15) than PPE17 (LRR 16–20) and facilitates its homodimerization to induce
IL-10 cytokine through activation of p38MAPK and, therefore, skews the T cell
response toward the Th2 type (Nair et al. 2009; Udgata et al. 2016). PPE18 simulta-
neously inhibits IL-12 and TNF-α pro-inflammatory cytokines by SOCS3–
p38MAPK–Rel signaling pathway (Nair et al. 2011). PPE37 reduces the level of
pro-inflammatory cytokines, i.e., TNF-α and IL-6, which is due to reduced level of
the phosphorylated p65 subunit of NF-κB, p38MAPK, and ERK1/2 (Daim et al.
2011). In an interesting study by Ahmad et al. (2018), it has been shown that N- and
C-terminal of PPE37 performs different functions in THP-1 macrophages. N-terminal
of recombinantly purified PPE37 causes proliferation and differentiation of mono-
cytic THP-1 into DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-
grabbing nonintegrin) which produces higher IL-10 than full length, whereas its
C-terminal localizes to macrophage nucleus and induces caspase-3-dependent apo-
ptosis. PE_PGRS33 and PE_PGRS26 were found to induce IL-10 in splenocytes
after infection when expressed in M. smegmatis and provided the survival advantage
to the bacteria (Singh et al. 2008). PE11 induces strong anti-inflammatory cytokines
which help the bacterium in progression of disease and persistence in a mouse model
of infection (Singh et al. 2016). Other than macrophages, dendritic cells (DCs) also
show a change in the cytokine profiles during M.tb infection. PPE34 facilitates DC
maturation via PI3K–MAPK and NF-κB signaling cascades and induces secretion of
high levels of Th2 cytokines (IL-10) when co-cultured with the CD4+ T cells (Bansal
et al. 2010). PPE38, when interacts with TLR2, induces both IL-6 and TNF-α in
THP-1 macrophages (Dong et al. 2012) and downregulates class I antigen presenta-
tion in peritoneal macrophage, thereby reducing/delaying the protective CD8+ T cell
activity (Meng et al. 2017). Khubaib et al. (2016) have shown that co-operonic PE32/
PPE65 shows greater modulation of immune responses in combination rather than
PE32 or PPE65 alone. Recombinant PE32 and PPE65 proteins together downregulate
TNF-α and IL-6 whereas upregulate IL-10 at higher concentrations in vitro indicating
a Th2-type response. In an in vivo experiment, they observed that recombinant
PE32 + PPE65 suppress IFN-γ in splenocytes and there was a reduction in IFN-γ-
and IL-2-positive CD4+ and CD8+ T cell populations.
8.4.1.3 Phagosome–Lysosome Fusion

Phagosome–lysosome membrane fusion is a highly regulated event that is essential
for intracellular killing of microorganisms. Phagosome maturation and acidification
is a process of internalizing particles and their trafficking into a series of increasingly
acidified membrane-bound structures, ultimately leading to particle degradation.
However, M.tb has evolved strategies to bypass this mechanism successfully. For
example, PE_PGRS62, when expressed in M. smegmatis, could arrest phagosome–
lysosome fusion by preventing the recruitment of Rab7 and LAMP1 to bacilli-
containing phagosomes (Thi et al. 2013). Similarly, a role of PPE54 in the preven-
tion of phagosome maturation was recorded (Brodin et al. 2010). BCG with four
pe_pgrs mutants (pe_pgrs5, pe_pgrs28, pe_pgrs44, and pe_pgrs59) and three
ppe_mptr mutants (ppe_mptr10, ppe_mptr16, and ppe_mptr21) were found to be
localized more predominantly in acidified phagosomes as compared to the wild-type
bacteria suggesting their roles in preventing phagosome maturation (Stewart et al.
2005). MAV-2928 (a homologue of PPE25) from M. avium was shown to prevent
the fusion of the acidified phagosome with lysosomes inside macrophages (Jha et al.
2010). PE_PGRS30 and its homologue in M. marinum, MAG24–1, have also been
found to interfere with phagosome maturation in macrophages via reduced recruit-
ment of LAMP1 (Iantomasi et al. 2012).
8.4.2 Adaptive Immune Responses
8.4.2.1 Antigen Presentation

Antigen presentation is a vital process in which antigen-presenting cells (APCs)
display processed antigens via major histocompatibility complex (MHC) which are
further recognized by subsets of T cells depending upon the type of MHC
molecules presenting the antigens. There are reports which state that PE_PGRS33
(Brennan and Delogu 2002) limits MHC class I antigen presentation. In a similar
study, Koh et al. (2009a) have shown that PE_PGRS17 inhibits antigen processing
by affecting ubiquitin–proteasome-dependent protein degradation and therefore
can prevent the killing of infected host cells. In a genome-wide screening, Saini
et al. (2016) have identified that PE_PGRS47 protein of M.tb is involved in
138 R. Pal et al.
inhibiting class II antigen presentation and autophagy in a nonredundant manner

by an unknown mechanism. In an unpublished study from our laboratory, PPE18
has also been shown to inhibit class II antigen presentation in vitro. Dendritic cells
(DCs) are one of the robust antigen-presenting cells. Some PE/PPE family proteins
have been shown to manipulate maturation of dendritic cells. For example, Bansal
et al. (2010) have shown that PPE34 possesses a SRC homology 3 (SH3) domain
which interacts specifically with TLR2 and elicits functional maturation of human
DCs. Similarly, PE25/PPE41 dimer is involved in maturation of mouse dendritic
cells (Chen et al. 2016).
8.4.2.2 B Cell and T Cell Responses

PE and PPE family members are a potentially rich source of B cell epitopes and
surface exposed, and a number of them are likely to generate humoral immune
responses. The highly repetitive domains of both PE and PPE proteins are
immunodominant. Ahmad et al. (2018) reveal that N- and C-terminal domains of
PPE37 perform different functions during infection conditions. Full-length and
N-terminal domain of PPE37 displayed stronger B cell response in TB patients
and immunized mice. Full-length PPE37 showed a higher IgG response in pulmo-
nary tuberculosis (PTB), extrapulmonary tuberculosis (EPTB), as well as patients
with relapse cases of TB, whereas N-terminal of PPE37 showed higher IgG
responses only in PTB cases (Ahmad et al. 2018). PPE57 has been shown to develop
a significant IgG response compared to ESAT-6 in sera of active TB and EPTB
patients as compared to healthy controls (Zhang et al. 2007). PE25 and PPE41 are
coded by a single operon. In sera obtained from TB patients, it was observed that
humoral response induced in TB patients against PE25/PPE41 complex was higher
than PPE41 and PE25 alone (Tundup et al. 2008). PPE42 a member of PPE_MPTR
class elicit humoral response primarily against domain rich in glycine and asparagine
(Chakhaiyar et al. 2004). Both recombinantly purified PPE44 and BCG infection
have shown antigen-specific IgG in mouse sera (Bonanni et al. 2005). The antibody
responses in TB patients were predominantly mounted against the N-terminal
domain of the PPE17 protein (Abraham et al. 2017). Co-operonic PE32 and
PPE65 together generate higher IgG1/IgG2a ratio and are known to support
enhanced survival of the bacilli (Khubaib et al. 2016).
T cell antigens have always been the center of attraction for development of
therapeutics as well as for mediating immunity that play leading roles in the control
of M.tb infections (Flynn et al. 1992; Caruso et al. 1999). Mycobacterium sp. have
been reported to elicit CD4+ and CD8+ responses (Jouanguy et al. 1999). Anti-
mycobacterial immunity is mediated by a Th1-type T cell. Th1 cells produce IFN-γ,
interleukin (IL)-2, and tumor necrosis factor (TNF)-β, which activate macrophages
and cell-mediated immunity. Studies have shown that PE/PPE proteins can be potent
T cell antigens. Among the antigens identified by peptide library screening for CD4+
T cell responses against M.tb, 45% were shown to be PE/PPE proteins. They
constitute more than 5% of the open reading frames of M.tb. The epitopes from
PE/PPE proteins constitute around 50% of the total pool of newly identified antigens
suggesting the potential of these proteins in regulation of host immune responses
(Kunnath-Velayudhan and Porcelli 2013) and therefore can be used to develop TB

vaccines. PPE44 lowers the IFN-γ in splenocytes after recombinant protein treat-
ment as well as with the BCG infection. Additionally, PPE44 modulates IgG1/IgG2a
ratio which skews the T cell response toward the Th2 type by producing higher
IgG1and low IFN-γ (Bonanni et al. 2005). PPE18 has also been shown to generate a
strong anti-inflammatory response by elevating IL-10 levels (Nair et al. 2009)
skewing the T cell response toward the Th2 type. PPE60 activates Th1/Th17
responses via Toll-like receptor 2-dependent maturation of dendritic cells (Su et al.
2018). PPE57 causes macrophage activation and drives Th1-type immune responses
through TLR2 (Xu et al. 2015). Balaji et al. (2007) have shown that PE_PGRS33
protein induces apoptosis in T cells in Smac-dependent activation of caspases. Choi
et al. (2018) have shown that when PE/PPE peptides were fused with ESAT-6, it
turned out to be a better T cell elicitor than ESAT-6 alone. In another study, it was
shown that pool of peptides from PE/PPE family elicits a higher IFN-γ response in
sensitized T cells obtained from both bovine and human hosts. It has been realized
that a subset of PE/PPE proteins are major targets of the cellular immune response to
tuberculosis and are recognized at multiple stages of infection and in different
disease states (Vordermeier et al. 2012). Another report by Chaitra et al. (2008a,
b) has shown that peptides derived from PE_PGRS33, PE_PGRS62, and PPE46
generate strong CTL response in PBMCs isolated from PPD-positive and TB
patients. The group showed that immunization with PE_PGRS62 or PPE46 DNA
construct induced CD8+ T cells and generated strong Th1-type response with high
IFN-γ and low IL-4 (Chaitra et al. 2008a, b).
All these studies indicate that PE/PPE proteins can modulate both innate and
adaptive immune responses of host and act as important virulence factors. Under-
standing individual and cumulative impact of PE/PPE proteins is an important aspect
which could provide the basis for novel interventions. However, the pathophysio-
logical functions of PE/PPE proteins are not fully understood. Therefore, further
approaches are needed to unravel the biological roles of these proteins for identifi-
cation of novel strategies to combat TB.
8.5 PE/PPE Proteins as Diagnostic Markers for Detecting TB

Cases
Early and accurate diagnosis of TB is one of the primary challenges in curtailing the
spread of TB. Current diagnosis of active pulmonary TB is mainly based on
confirmation of acid fast bacilli by sputum smear microscopy and culture of
mycobacteria. But sputum smear examination displays a low degree of sensitivity
which is about 50–60%, and the sensitivity is further reduced especially in children
and patients coinfected with HIV. Mycobacterial culture, a gold standard method of
TB diagnosis, is time-consuming, and more importantly, TB bacilli fail to grow in
10–20% of positive cases (Andersen et al. 2000). Using conventional diagnostic
method, it is cumbersome to diagnose both extrapulmonary and smear-negative
TB. It is estimated that 13–17% of newer infections are caused by smear-negative
140 R. Pal et al.
TB (Tostmann et al. 2008). WHO recommended rapid and fully automated Xpert
MTB/RIF test for the diagnosis of TB in children and some forms of extrapulmonary
TB (WHO 2014). Although molecular diagnostic tools like polymerase chain
reaction (PCR) are rapid, highly sensitive, and specific for the diagnosis of different
forms of TB, their application is limited due to higher cost, availability of reagents,
requirement of technical expertise, and poor specificity under field conditions.
Serological assays that measure antibody responses to selective M.tb antigens in
suspected cases of TB are still considered to be attractive tools due to simplicity and
convenient detection of pathogens, low costs, easy operation, and rapid determina-
tion. According to recommendations of the WHO in order to replace the “gold
standard” sputum culture, a serological test for active TB should have sensitivities of
over 80% and test specificities of over 95% (WHO 2014). Recently WHO has
estimated that more than 50 companies are involved in the development of rapid
TB diagnostic tests (WHO 2014) and globally several attempts are being made to
identify and test the serodiagnostic potential of various M.tb antigens (Baumann
et al. 2014; Khurshid et al. 2014; Pukazhvanthen et al. 2014; Feng et al. 2013; Zhang
et al. 2013).
Several groups have reported higher sensitivity of PPE proteins to detect active
TB patients. Examples are PPE2 (Abraham et al. 2014), PPE17 (Abraham et al.
2016; Khan et al. 2008), PPE41 (Choudhary et al. 2003), PPE42 (Ireton et al. 2010;
Chakhaiyar et al. 2004), PPE44 (Rindi et al. 2007), PPE57 (Zhang et al. 2007),
PPE65 (He et al. 2011), and PPE68 (Xu et al. 2012) for diagnosis of active
TB. Tundup et al. (2008) reported PPE41 was sensitive in detecting 45% of TB
cases; however, its sensitivity was increased to 75% when the antigen was combined
with a PE protein (PE25/PPE41 protein complex). Khan et al. (2008) showed that
PPE17 could strongly discriminate active TB patients from BCG-vaccinated healthy
controls compared to PPD, ESAT-6, and HSP60. PPE17 showed 81.3% sensitivity
toward extrapulmonary TB. The serodiagnostic potential of PPE17 was also studied
in smear-negative TB cases, and interestingly, PPE17 displayed higher sensitivity
(75%) for this category of TB as well. In sera derived from TB patients, PPE2 protein
too generates higher titer of IgG than PPD when compared with BCG-vaccinated
healthy controls (Abraham et al. 2014). Though PPE2 could detect smear-negative
and extrapulmonary TB patients, it is less sensitive than PPE17 (Abraham et al.
2016). PPE17 is surface exposed (Donà et al. 2013), and genes that are homologous
to PPE17 are absent in the nontuberculous mycobacterial species. PPE17 is shown to
be upregulated in conditions that mimic the macrophage environment features.
Moreover, overexpression of PPE17 protein was observed in macrophages infected
with various clinical isolates of M.tb (Homolka et al. 2010). All these features
suggest that PPE17 could be a potential serodiagnostic marker for detecting active
TB cases. Abraham et al. (2017) reported that the N-terminal domain of PPE17
protein plays a dominant role in inducing antibody responses in active TB patients
including the smear-negative and extrapulmonary TB. Although the N-terminal
domain of PPE proteins is conserved, PPE17 showed a unique amino acid sequence
starting from 122 amino acid to 140 amino acid (IFGIHTPAIFALDALYAQY) in its
N-terminal region and did not significantly cross-react with N-terminal domains of
other PPE proteins such as PPE18, PPE44, and PPE65 (Abraham et al. 2017).
Tundup et al. (2008) reported that PE25 (Rv2431c) has very low sensitivity as it
could detect only 9% of TB cases (n ¼ 32). Another group demonstrated that among
the three PE antigens PE11, PE_PGRS17, and PE_PGRS33, PE_PGRS17 showed
higher sensitivity toward newer and extrapulmonary TB patients (Narayana et al.
2007). Baumann et al. (2014) compared PE35 with other M.tb antigens and observed
that PE35 could not display statistically significant IgG response, but showed higher
IgA response (78.6%). Interestingly, Mukherjee et al. (2007) reported that PE35
displayed a higher sensitivity of 92.5% and 88.8% against pulmonary and
extrapulmonary TB patients diagnosed in Kolkata, India.
PE/PPE proteins have been studied for serological diagnosis of latent TB as well.
Koh et al. (2009b) studied the seroreactivity to PE-PGRS17 and PE-PGRS62
proteins and observed that PE-PGRS62 is associated with both latent and active
TB. PPE44 showed a sensitivity of 37.5% in latent TB infection (Rindi et al. 2007).
One of the highly immunogenic PPE protein, PPE55, was showed to be useful to
differentiate between latent TB and incipient, subclinical TB (Singh et al. 2005). A
recent study showed PPE68 could be used as a sensitive and specific biomarker for
discriminating TB from LTBI (Pourakbari et al. 2015). An interesting study reveals
that PPE17 could discriminate individuals with latent TB infection from the
QFT-negative subjects and demonstrated a higher sensitivity (87%) for the detection
of latent TB-positive individuals (Abraham et al. 2018).
Other than serological assays, Srivastava et al. (2006) employed PCR for ampli-
fication of 1291 bp fragment of M.tb that belongs to a PPE gene family, Rv0355
(PPE8). Interestingly, PCR was found to be highly sensitive for detection of M.tb in
sputum, CSF, and pleural fluids (90.4%, 70%, and 77.7%, respectively) compared to
the culture method (62.9%, 10%, and 24.4%, respectively). Recently Yuan and Xu
(2017) screened genes of M.tb with comparative genomics approach and found that
PPE66 (Rv3738c) can be amplified in clinical samples. However, this was a
preliminary study with the sample size of only 15 and needs further detail testing.
Overall, with the limitations of conventional and existing diagnostic methods, it is
cumbersome to diagnose active TB especially smear-negative, extrapulmonary, and
latent TB infection. Thus, PE/PPE proteins can be used as potential markers for
serodiagnosis of active TB especially smear-negative, extrapulmonary, and latent
tuberculosis infection.
8.6 PE and PPE Proteins as Vaccine Candidates
Since 1940, BCG is the only vaccine against tuberculosis used around the globe. But
partial effectiveness of BCG in demographically distinct populations and prevalence
of MDR and XDR incidences demand designing of novel TB vaccines. There could
be three strategies to develop new TB vaccines like i) improvement of existing BCG
by adding immunogenic antigens, ii) creating more attenuated M.tb stains by genetic
manipulation, and iii) use of DNA or protein subunits for boosting the immune
responses. Being a potent T cell antigen, PE and PPE proteins can be promising
142 R. Pal et al.
candidates for TB vaccine. There are various preliminary reports stating the protec-
tive role of PE and PPE proteins in an animal model of infection. Delogu and
Brennan (2001) have shown that PE_PGRS33 produces a prophylactic effect in
the mouse as both DNA and protein subunit. In another study, PE4 was shown to act
as a potent protective antigen (Singh et al. 2013a, b). Similarly, increased protection
against M.tb was recorded during immunization with PPE14 (MTB41) in both
mouse and guinea pig model (Skeiky et al. 2005). PPE57 protein is considered to
be a potential antigen for the design of a vaccine against M.tb (Xu et al. 2015). Also,
it has been noticed that immunization of mice with recombinant PE3 protein induced
a strong protective immune response against challenge with live mycobacteria and
can be used as a candidate to develop subunit vaccines (Singh et al. 2013a, b).
Similarly, PPE15 was shown to induce significant protection when administered
alone and as a boost to BCG vaccination in mice (Stylianou et al. 2018). There are
studies which show improved efficacy of polyprotein subunit candidates, when
fused with PE and PPE proteins. The fusion of MPT65 with PE domain of
PE_PGRS33 (cell wall-localized protein) results in localization of MPT65 antigen
on the surface of BCG and enhances the efficiency of BCG vaccine against M.tb
infection in mice (Sali et al. 2010). Further, deletion of PE and PPE genes of ESX-5
caused the reduction in M.tb virulence in immunocompetent mice (Sayes et al.
2012). This M.tb mutant (Δppe25-pe19) strain was highly attenuated and was able
to generate T cell response against the major ESX-1 virulence factors (ESAT-6,
CFP-10) and other ESX-associated PE and PPE proteins (Sayes et al. 2012). Another
study by Skeiky et al. (2004) revealed that immunization of C57BL/6 mice with
Mtb72F (a 72 kDa polyprotein harboring Mtb32C-Mtb39 (PPE18)-Mtb32N in linear
order) resulted in protection against M.tb infection. However, there are clinical
strains which have variations in PPE18 region, which can make Mtb72F less
efficient. ID93, another vaccine candidate, contains PPE42 as one of the components
of 93 kDa polyprotein complex (Rv3619-Rv1813-Rv3620-Rv2608/PPE42). This
polyprotein subunit vaccine along with adjuvant has been shown to provide protec-
tion against drug-resistant M.tb in mice and guinea pig and therefore can be
considered as the candidate molecule for improving the efficacy of the existing
BCG vaccines (Bertholet et al. 2010).
Being a potent T cell antigen, PE/PPE proteins showcase themselves as a good
candidate for vaccine development. Therefore, further research is needed to uncover
the potential of PE/PPE proteins for improving the efficacy of existing vaccines.
8.7 Conclusion
M.tb has coevolved a long way along with the host and has adopted several strategies
to evade host immune responses. MDR and XDR are posing new challenges in the
field of TB vaccination and treatment. Therefore, detailed knowledge of “factors”
that play crucial roles in the pathophysiology of the bacilli is needed to design better
therapeutics to fight against this deadly disease. In a comparative genomic analysis
of H37Rv and H37Ra, Kohli et al. (2012) have reported several key changes in the
nucleotide sequences among PE and PPE homologues. These changes in the genes
are often believed to be the cause of major or physiochemical changes in the encoded
proteins, therefore affecting virulence. Further, physiological and biochemical
assays are needed to understand the implication of these genetic variations. More-
over, presence of PE and PPE proteins only in the pathogenic species is a strong
indicator of their roles in M.tb pathogenesis. Therefore, extensive studies are
required to comprehend the role of PE and PPE proteins in host immunomodulation
and then further expand it to understand the mechanisms of virulence, pathogenesis,
and latency of TB. Detailed immunological studies of PE and PPE proteins might
help to utilize them as therapeutics to treat other pathological conditions. For
example, a novel role of PPE18 as an anti-sepsis molecule is established in mouse
model of E. coli-induced and CLP-induced septicemia (Ahmed et al. 2018).
Acknowledgments The authors thank Dr. Philip Raj Abraham for his valuable suggestion in
preparing the manuscript. The laboratory of SM is supported by the grants from the Department of
Biotechnology (DBT), Govt. of India (BT/HRD/35/01/03/2018 and BT/PR12817/COE/34/23/
2015), and Department of Science and Technology-Science and Engineering Research Board
(DST), Govt. of India (EMR/2016/000644), and also core grant from CDFD by DBT. RP is
supported by a fellowship from the Department of Biotechnology, Govt. of India.
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Intrinsically Disordered Regions in PE/PPE
Protein Family of Mycobacterium 9
tuberculosis: Moonlighting Function
Farha Naz, Javeed Ahmad, Mohd Shariq, Mohd Arish,

Javaid A. Sheikh, Seyed E. Hasnain, and Nasreen Z. Ehtesham
Abstract
Mycobacterium tuberculosis, the causative agent of tuberculosis, has the distinc-
tion of harboring an unusual set of plentiful antigens that are highly homologous.
The abundance of similar proteins like PE/PPE that are restricted to pathogenic
mycobacterium species implies their important role in either homeostasis or
helping to adapt to the intracellular niche which is one of the major life cycle
stages in pathogenesis of this bacterium. In spite of the various important
functions delineated for this family, it is always challenging to study this
extremely homologous family of proteins due to difficulties associated with
expression and purification. Phylogenetic studies suggest that pe/ppe genes
coevolved with esx loci, which encodes proteins that form secretion systems for
the export of PE/PPE proteins. Despite the biochemical and immunological
characterization of various PE/PPE proteins, there is dearth of structural data
for PE/PPE complex. The structural details and thus the structure-function rela-
tion are essential to understand the utility of homologous protein repertoire in
pathogenesis. Moreover, the atomic detail could unravel the novel targets to curb
F. Naz · J. Ahmad · M. Shariq · N. Z. Ehtesham (*)
M. Arish
J. A. Sheikh
Department of Biotechnology, School of Chemical and Life Sciences, Jamia Hamdard, New Delhi,
Delhi, India
S. E. Hasnain

https://doi.org/10.1007/978-981-32-9413-4_9
152 F. Naz et al.
this obnoxious pathogen. This chapter will present in detail the structural organi-
zation as well as the disorder content of the PE/PPE family. This structural
enigma of being highly homologous along with having highest disorder content
will be explained with possible functional significance. The available crystal
structure and predictions on the basis of those accompanied with functional
aspect of their role in cell death pathways are also detailed herein.
Keywords
Structural disorder · PE/PPE proteins · Crystal structure · Programmed cell death ·
Apoptosis · Necrosis
Abbreviations
(M.tb) Mycobacterium tuberculosis

LCR Low complexity region
PCD Programmed cell death
PE Proline-glutamic acid
PPE Proline-proline-glutamic acid
RMSD Root-mean-square deviation
TB Tuberculosis
9.1 Introduction
Mycobacterium tuberculosis(M.tb) causes tuberculosis (TB), one of the most

devastating diseases worldwide, and approximately kills two million people annu-
ally (Glaziou et al. 2013). M.tb has evolved from being nonpathogenic and attained
pathogenicity during coevolution with humans. During the course of evolution,
genome size has decreased (Rahman et al. 2014). The only exception to this
prodigy is family of methyltransferase proteins and PE/PPE protein family (Gey
van Pittius et al. 2006). The characteristic feature of the PE family is sequence of
amino acids (proline-glutamic acid (PE) at eighth and ninth position) in a
conserved N terminal domain of about 110 amino acids. PPE family also has a
highly conserved N terminal but extended to about 180 amino acids and has the
presence of proline-proline-glutamic acid (PPE) at 7th, 8th, and ninth position,
respectively. Despite being highly conserved, there is no homology in this N
terminus region between PE and PPE. The C terminals of the both PE/PPE protein
families are of variable size with other distinctive features (Cole 1999). Although
the precise function for this family of proteins is yet to be established, the fact that
this family encodes about 4% proteins and has 10% of genome dedicated to it
suggests some vital functions. The polymorphic nature of C terminal regions
suggests their role in antigenic variation (Cole 1999). These proteins are involved
in immune subversion as these proteins are known to interact and diminish the
9 Moonlighting Function of PE/PPE Proteins 153
antigen processing and presentation (Delogu and Brennan 2001; Khubaib et al.
2016). Emerging evidence has implicated these proteins in a wide array of roles
(Brennan 2017). Functional studies have validated that PE/PPE proteins localize to
the membrane, thus forming an integral part of secretion systems. These proteins
are associated with type VII mycobacterial secretion systems that are vital for
pathogenesis of mycobacteria. This family also encodes lipolytic enzymes, and
these enzymes form major virulence factors in mycobacterial metabolome. The
evidence of their cell wall localization implicate that these proteins may directly
interact with the host and be possibly involved in the modulation of innate as well
as adaptive immune functions which makes this family of proteins highly desirable
to be evaluated as prophylactic and therapeutic targets. Apart from immune
modulatory strategies, proteins involved in the crucial pathways could be exploited
as novel drug targets. However, exploring this promising family involves various
challenges due to extensive homology and repetitive sequences. The functional
redundancies due to the similarity do not allow ascertaining the functionality to a
protein very precisely. Similar reasons impact the determining of crystal structure
of these proteins that eventually affects elucidation of structure-function relation-
ship. The limited crystal structures available limit the exploration of these ambig-
uous proteins, which is one of the major obstacles in determining the functional
significance of the PE/PPE proteins. More verified crystal structures and stringent
modeling approaches will help in determining an integrated view of the
interactome of this protein family. This will also aid in the development of novel
therapeutic approaches and interventions against this deadly pathogen (Fishbein
et al. 2015).
The ability of these proteins to arm mycobacteria with sophisticated pathways to
modulate host cell programmed death is very intriguing. This interplay of host-
pathogen interaction is extremely vital for understanding the pathogenesis of tuber-
culosis. Unraveling the mechanism of modulating host cell death pathway could
enable us to develop therapeutic interventions. Moreover, better understanding of
pro-survival and pro-death factors could give insights into developing novel and
efficacious vaccines which is the need of hour.
This chapter will familiarize the readers with structural features and some func-
tional aspects of PE/PPE proteins. The structural disorder in this protein family and
the information from the limited crystals are elucidated in detail. Moreover, the
modulation of cell death pathways by these proteins will highlight the decoy
mechanisms employed by this resilient pathogen.
9.2 Structural Organization of PE/PPE Operons: Functional

Relevance
The PE proteins have conserved Pro-Glu (PE), and PPE have Pro-Pro-Glu (PPE)
motifs at their N terminal end of the proteins. PE/PPE protein family accounts for
about 10% of the coding capacity of the M.tb genome (Cole et al. 1998). The
respective genes are systematized in a definite operonic pattern within the genome,
154 F. Naz et al.
where a pe gene is followed by a ppe gene (Tundup et al. 2006). The members of
PE/PPE protein family are usually associated with members of the ESX family
proteins that are important virulence factors, T cell antigens, and potential subunit
vaccine candidates. Almost all of the PE/PPE family proteins probably evolved from
the duplication of the ancestral esx-related pe/ppe genes, and they are significantly
expanded in the slow-growing mycobacteria (Brennan and Delogu 2002; Cole et al.
1998). However, the functions of most of the PE/PPE proteins remain to be
deciphered, and few of them have been shown to play important roles in mycobac-
terial virulence, required for growth of mycobacteria inside the macrophages or in
the mouse infection model, or play important role in the inhibition of phagosome
maturation of infected macrophages (Bottai and Brosch 2009; Bottai et al. 2012;
Brennan et al. 2001; Brodin et al. 2010; Dong et al. 2012; Gey van Pittius et al. 2006;
Goldstone et al. 2009; Iantomasi et al. 2012; Li et al. 2005; Sampson 2011; Sampson
et al. 2001). PE/PPE proteins exhibit number of repetitive sequences and harbor
abundant immunogenic regions, forming a rich source of B and T cell epitopes
(Copin et al. 2014). The esx-5 region of M.tb (rv1782-rv1798) comprises two pe
(pe18, pe19) and three ppe (ppe25, ppe26, ppe27) genes. These PE and PPE proteins
are secreted through the transmembrane channel formed by the ESX-5 secretion
apparatus involving the ESX conserved essential component EccD5 (Bottai et al.
2012). In addition, many of the other PE/PPE proteins not associated with esx-5
locus but having certain degree of sequence similarity with their esx-5 coded
counterparts are also secreted via the ESX-5 secretion system (Abdallah et al.
2006, 2009; Majlessi et al. 2015). Earlier, it was assumed that PE, PPE, and EspG
proteins that belong to a particular esx loci tend to interact with each other only and
associate with other components of the same cluster, but recent evidence suggests
that there may be some cross talk involved. This remains to be elucidated whether
this cross talk has any functional relevance or it is just one esx loci compensating for
the missing components of other esx loci.
There are ~99 PE and ~69 PPE proteins with ~100 and ~180 conserved N
terminal residues, respectively, in the genome of M.tb (Brennan 2017). Among
them, 61 falls in PE/PGRS (polymorphic GC-rich sequence) and the rest 38 belongs
to PE only (Poulet and Cole 1995; Zumbo et al. 2013). Numbers can vary for
different strains of M.tb. The PE/PGRS contributes significant role in pathogenesis
of M.tb. The pe/pgrs genes contain significantly higher nucleotide diversity within
the members of this family. The gene of PE/PGRS family proteins is mostly
clustered in a region of M.tb genome as overlapping genes. To maintain the different
functions in mycobacterial virulence, PPE proteins diversify to PPE/MPTR (major
polymorphic tandem repeats), PPE/SVP (Gxx-SVPxxW motif), and PPE/PPW
(PxxPxxW motif) subgroups (Fishbein et al. 2015). The PPE proteins play a key
role in secretion and localization of different proteins (Daleke et al. 2011; Dona et al.
2013). All genes are scattered throughout the chromosome, but pe/ppe genes are
found to be located in the vicinity of each other. There are certain pairs of pe/ppe
genes which are found to be co-localized with each other, and these are pe1 and pe2,
pe5 and ppe4, pe/pgrs12 and pe_pgrs13, pe7 and ppe14, pe9 and pe10, pe11 and
ppe17, pe15 and ppe20, pe18 and ppe26, pe20 and ppe31, pe22 and ppe36, pe25 and
ppe41, pe29, ppe47 and ppe48, ppe50 and ppe51, ppe54, pe/pgrs49 and pe/pgrs56,
pe32 and ppe65, pe/pgrs60 and pe/pgrs61, ppe66 and ppe67, and pe35 and ppe68
(Table 9.1). There are approximately 22 predicted operonic clusters out of 69 PPE
proteins (Akhter et al. 2012; Tundup et al. 2008). Many among these are transcribed
and function together as there is less than 100 bp gap between them in 14 of the gene
pairs. Some of these are secreted proteins or present on the cell surface together as
heterodimers. pe5/ppe4 and pe15/ppe20 have only 2 bp and 4 bp intergenic
distances, respectively, between their pe/ppe genes in M.tb and are co-operonic
too, but they don’t show any physical interaction between their PE/PPE proteins
as the gene pair pe25/ppe41, which is co-operonic and shows interaction with each
other to form stable protein complex (Tiwari et al. 2012).
9.3 Structural Disorder in PE/PPE: The Molecular Enigma
The molecular function of a protein is fundamentally related to its three-dimensional

structure. This hypothesis laid down the foundation of sequence-structure-function
paradigm. It is well-known datum that most of the proteins and polypeptide
segments fold co-operatively into well-defined three-dimensional structure. How-
ever, earlier interpretation that proteins possess an absolute functional specificity
with fixed structure disagrees with their marked ability to evolve and adapt new
structures and functions. There is growing evidence from last few decades that
suggests protein structural flexibility due to structural disorder plays an important
role in many different biological systems. Many experimental evidences also suggest
that disordered regions in proteins are involved in many types of molecular
recognitions. One of the advantages of disordered binding sites is to recognize
several targets with low affinity and high specificity allowed by their multiple
meta-stable conformations (Dunker et al. 2001; Wright and Dyson 1999).
Transitions between the native unfolded state and a globular structure induced by
some type of interactions may also provide thermodynamic regulation of binding.
These studies are now establishing the disorder-function paradigm. Such proteins are
known as intrinsically disordered or unstructured proteins (Dunker et al. 2001). It
has been shown in a number of studies that there are clear patterns that characterize
disordered regions such as low sequence complexity regions and amino acid com-
position bias and, therefore, can be predicted successfully from amino acid sequence
(Dunker et al. 2001; Iqbal and Hoque 2016).
Computational studies from our laboratory and other groups disclosed PE/PPE
protein family is structurally highly disordered compared to the rest of the proteome
of M.tb (unpublished data). The relatively high level of structural disorder signifies
their important role and supports their anticipated functions in immune evasion and
host-pathogen interactions, since many known pathogenic effector proteins are
disordered. Amino acid composition analysis of PE/PPE protein family is given in
Fig. 9.1. Moreover, the pathogenesis-related PE/PPE/PGRS family is highly
enriched with glycine- and alanine-rich low complexity regions (Table 9.2).
Table 9.1 Co-operonic PE/PPE genes: PE and PPE genes generally exist in single operon, but some of them found to be present alone
156
Operon GI Synonym Gene Start End Strand Length Product

6924 57116694 Rv0151c pe1 177,543 179,309 - 588 PE family protein
57116695 Rv0152c pe2 179,319 180,896 - 525 PE family protein
6950 57116714 Rv0280 ppe3 339,364 340,974 + 536 PPE family protein
15607422 Rv0281 340,998 341,906 + 302 Hypothetical protein
6951 15607423 Rv0282 342,130 344,025 + 631 Hypothetical protein
57116715 Rv0285 pe5 349,624 349,932 + 102
PE family protein
57116716 Rv0286 ppe4 349,935 351,476 + 513
15607428 Rv0287 esxG 351,525 351,818 + 97 PPE family protein
15607429 Rv0288 esxH 351,848 352,138 + 96 Hypothetical protein
15607430 Rv0289 352,149 353,036 + 295 Low-molecular-weight protein antigen
15607431 Rv0290 353,083 354,501 + 472 7 ESXH (10 kDa antigen) (CFP-7)
15607432 Rv0291 mycP3 354,498 355,883 + 461 (protein TB10.4)
Transmembrane protein
Membrane-anchored mycosin
Transmembrane protein
533901 224988670 JTY_0292 343,390 345,285 + 631 Hypothetical protein
Mycobacterium 224988671 JTY_0293 345,282 346,898 + 538 Hypothetical protein
bovis BCG str. 224988672 JTY_0294 346,895 350,887 + 1330 Hypothetical protein
Tokyo 172 224988673 JTY_0295 pe5 350,884 351,192 + 102
PE family protein
224988674 JTY_0296 ppe4 351,195 352,736 + 513
224988675 JTY_0297 esxG 352,785 353,078 + 97 PPE family protein
224988676 JTY_0298 esxH 353,108 353,398 + 96 Hypothetical protein
224988677 JTY_0299 353,409 354,296 + 295 Low-molecular-weight protein antigen
224988678 JTY_0300 354,343 355,761 + 472 7
Hypothetical protein
F. Naz et al.
9
224988679 JTY_0301 355,758 357,143 + 461 Putative transmembrane protein

224988680 JTY_0302 357,140 358,135 + 331 Putative protease precursor
Putative transmembrane protein
260342 121636197 BCG_0322 373,058 374,953 + 631 Hypothetical protein
121636198 BCG_0323 374,950 376,566 + 538 Hypothetical protein
121636200 BCG_0325 pe5 380,552 380,860 + 102
PE family protein
121636201 BCG_0326 ppe4 380,863 382,404 + 513
121636202 BCG_0327 esxG 382,453 382,746 + 97 PPE family protein
121636203 BCG_0328 esxH 382,776 383,066 + 96 Hypothetical protein
121636204 BCG_0329 383,077 383,964 + 295 Low-molecular-weight protein antigen
121636205 BCG_0330 384,011 385,429 + 472 7 CFP-7
121636207 BCG_0332 386,808 387,803 + 331
Moonlighting Function of PE/PPE Proteins
Putative protease precursor

6972 15607528 Rv0387c 466,672 467,406 244 Conserved hypothetical protein
57116729 Rv0388c ppe9 467,459 468,001 180 PPE family protein
6986 15607582 Rv0441c 530,296 530,724 142 Hypothetical protein
57116731 Rv0442c ppe10 530,751 532,214 487 PPE family protein
7057 15607885 Rv0745 835,154 835,681 + 175 Hypothetical protein
57116773 Rv0746 pe_pgrs9 835,701 838,052 + 783 PE-PGRS family protein
7079 57116787 Rv0832 pe_pgrs12 924,951 925,364 + 137 PE-PGRS family protein
57116788 Rv0833 pe_pgrs13 925,361 927,610 + 749 PE-PGRS family protein
7098 57116795 Rv0915c ppe14 1,020,058 1,021,329 423 PPE family protein
57116796 Rv0916c pe7 1,021,344 1,021,643 99 PE family protein
7138 57116823 Rv1088 pe9 1,214,513 1,214,947 + 144 PE family protein
57116824 Rv1089 pe10 1,214,769 1,215,131 + 120 PE family protein
157
(continued)
158
Operon GI Synonym Gene Start End Strand Length Product

57116837 Rv1169c pe11 1,299,822 1,300,124 100 PE family protein
57116858 Rv1387 ppe20 1,561,769 1,563,388 + 539 PPE family protein
7538 57117029 Rv2853 pe_pgrs48 3,162,268 3,164,115 + 615 PE-PGRS family protein
15609991 Rv2854 3,164,152 3,165,192 + 346 Hypothetical protein
57117030 Rv2855 mtr 3,165,20 3,166,5 + 459 Mycothione reductase
15609993 Rv2856 nict 53,166,684 843,167,802 + 372 Nickel-transport integral membrane
protein
7577 15610156 Rv3019c esxR 3,378,711 3,379,001 96 Secreted ESAT-6 like protein ESXR
57117047 Rv3020c esxS 3,379,036 3,379,329 97 (TB10.3) (ESAT-6 like protein 9)
57117048 Rv3021c ppe47 3,379,376 3,380,452 358 Esat-6 like protein esxS
57117049 Rv3022c ppe48 3,380,440 3,380,682 80 PPE family protein
57117050 Rv3022A pe29 3,380,679 3,380,993 104 PPE family protein
PE family protein
7599 57117064 Rv3135 ppe50 3,501,334 3,501,732 + 132 PPE family protein
57117093 Rv3344c pe_pgrs49 3,736,984 3,738,438 484 PE-PGRS family protein
57117094 Rv3345c pe_pgrs50 3,738,158 3,742,774 1538 PE-PGRS family protein
F. Naz et al.
9
7698 57117116 Rv3511 pe_pgrs55 3,939,617 3,941,761 + 714 PE-PGRS family protein
57117117 Rv3512 pe_pgrs56 3,941,724 3,944,963 + 1079 PE-PGRS family protein
7722 15610755 Rv3619c esxV 4,059,984 4,060,268 94 ESAT-6 like protein ESXV (ESAT-6
15610756 Rv3620c esxW 4,060,295 4,060,591 98 like protein 1)
57117135 Rv3621c ppe65 4,060,648 4,061,889 413 ESAT-6 like protein ESXW (ESAT-6
57117136 Rv3622c pe32 4,061,899 4,062,198 99 like protein 10)
PPE family protein
PE family protein
7727 57117139 Rv3652 pe_pgrs60 4,093,632 4,093,946 + 104 PE-PGRS family-related protein
57117140 Rv3653 pe_pgrs61 4,093,940 4,094,527 + 195 PE-PGRS family-related protein
57117151 Rv3739c ppe67 4,190,284 4,190,517 77 PPE family protein
7778 57117163 Rv3872 pe35 4,350,745 4,351,044 + 99 PE family-related protein
Moonlighting Function of PE/PPE Proteins
159
160 F. Naz et al.
Fig. 9.1 Amino acid composition analysis of PE/PPE protein family: PE/PPE proteins are glycine
and alanine rich. PE_PGRS has more number of glycine residues
Table 9.2 PE/PPE protein family is glycine and alanine rich and enriched with low complexity
regions
Alanine-rich LCRs Glycine-rich LCRs
Protein family Terminal LCRs Central LCRs Terminal LCRs Central LCRs
PE 26 1
PE/PGRS 57 4 57
PPE 58 11 2 22
The high structural disorder might also be the reason for difficult expression,
purification, and crystallization of these proteins. Therefore, only few pairs have
been crystallized till date (Chen et al. 2017; Ekiert and Cox 2014; Strong et al. 2006).
The individual members of PE/PPE protein family are structurally more disordered
and undergo mutual folding-upon-binding to attain higher structural order. Thus, the
observed structural disorder in the free forms of PE and PPE domains and following
a mutual folding-upon-binding mechanism of interaction could allow for faster
evolutionary rates (unpublished data). These strategies might enable the pathogen
to efficiently develop novel cognate pairs of PE/PPE proteins that might be crucial
for its adaptive survival. Interestingly, the high structural disorder in PE/PPE protein
family was observed mostly in variable C terminal regions which likely contain short
linear interaction motifs embedded into disordered regions in some subfamily
members (unpublished data) and may be more extended, domain-like interaction
modules in some others (Ahmad et al. 2018).
The high structure disorder and expansion of PE/PPE protein family in patho-
genic species implicate counterbalancing of reductive evolution by employing this
specific PE/PPE family of proteins by M.tb. Many members of PE/PPE protein
family accomplish structure ordered state through protein-protein interactions or
retain partial disordered states even after the formation of the specific complex.
Numerous members of PE/PPE protein family are membrane localized and surface
exposed showing high levels of functional specialization among family members
through disordered and variable C terminal. PE/PPE protein complexes successfully
impact the host immune system favoring pathogen survival through pro-pathogen
immune response.
9.4 Crystal Structure of PE/PPE Proteins: The Way Forward
The expression of individual PE or PPE proteins is difficult, and they need each other
to get folded (Strong et al. 2006). But, the expression and purification of individual
PE5, PE15, PE32, and PPE65 proteins have been done (Khubaib et al. 2016; Tiwari
et al. 2012). The structures of these PE/PPE proteins are not so clear because of the
unavailability of crystal structures. There is no crystal known for individual PE/PPE
protein till now. Only two PE/PPE pairs have been crystallized so far. The structures
of the two known PE/PPE pairs are shown in Fig. 9.2.
The complex is heterodimeric and consists mainly of α-helix. It has one PE and
one PPE protein. The PE protein consists of two-helix bundle and PPE protein has
five helices. The two α-helices of PE interact with two α-helices of PPE protein to
form four-helix bundle.
Fig. 9.2 3-D structure of PE/PPE proteins: PE proteins are represented in red; PPE proteins are
shown in blue color. PE/PPE proteins are helix-rich proteins with number of interacting residues
between them
162 F. Naz et al.
9.5 Structural Organization of PE25/PPE41
The PE25 protein has 99 amino acids, along with two α-helices, and the PE motif,
residues 8–9, is found to be present in the electron density map at N terminal of the
protein. The residues 8–37 form one α-helix, and residues 45–84 form another
α-helix of PE25. Both the helices are antiparallel with each other and connected
by residues 38–44 forming loop. N and C termini of the protein are located at the top
of the complex. Both the helices of PE protein interact with helices 2 and 3 of the
PPE protein, and loop in between the helix of PE protein interacts with helices 2 and
5 of the PPE protein to make the PE protein stabilized. Due to the sequence
similarities, nearly all the PE proteins of M.tb share similar structural organization.
The PPE41 protein has 194 amino acids, along with five α-helices, and the PPE
motif, residues 7–9, is found to be present in the electron density map at N terminal
“hook” of the protein. This hook protects the interacting PE protein. Extensive polar
regions, residues 21–53, form helix 2, and residues 58–103 form helix 3 of PPE
proteins which run antiparallel with each other and do interact with PE protein to
make several hydrophobic and stearic interactions.
There are basically four regions of conserved residues, and the first high conser-
vation region lies in the interior of four-helix bundle at the interface of PPE with PE
protein and is conserved in many PE/PPE pairs due to sequence similarities. The
second high conservation region lies in the PE loop that interacts with the PPE
protein surface. The third high conservation region includes the PPE sequence motif,
and the fourth high conservation region is in the C terminus of the PPE protein, a
poly-proline-rich region. The tyrosine (Y139) of PPE and proline (P170, P171,
P172, and P70) of the PE are one of the most conserved residues.
9.6 Structural Organization of PE8/PPE15
The purification of individual PE8 and PPE15 protein was difficult, so truncated
PE81–99 and PPE151–194 were purified together. To get the crystal of these proteins,
EspG5, a specific chaperone for PE/PPE proteins was included. All the three proteins
exist in a stoichiometric ratio of 1:1:1 and form an elongated-shaped structure in
solution (Chen et al. 2017). Residues 85–99 of PE8 (YxxxD/E secretion motif) and
residues 174–194 of PPE15 are highly disordered. So the crystal structure which is
known consists of 7–299 of EspG5, residues 7–84 of PE8, and residues 1–173 of
PPE15. The structure of PE8/PPE15 is found to be similar to PE25/PPE41 with
RMSD of 2.475 Å (PE) and 2.037 Å (PPE). The overall RMSD is calculated to be
1.9. Figure 9.3 EspG5 interacts only with helix 4 (α-4) and helix 5 (α-5) of PPE15
just at the opposite end of the PE/PPE heterodimer. The two helices of PE8 interact
with the α-1, α-2, α-3, and α-5 of PPE15 to form a four-helix bundle.
The C terminal YxxxD/E secretion motif of PE and WxG motif of PPE in
PE8/PPE15 and PE25/PPE41 are in different orientation. In PE25/PPE41, both the
motifs are found to be in close proximity and form Van der Waals contacts between
Y87 of PE25 and W56 of PPE41, whereas, in case of PE8/PPE15, there is no
Fig. 9.3 Superimposed

image of PE25/PPE41 and
PE8/PPE15 proteins with 1.9
RMSD. Red, PE25; raspberry,
PE8; blue, PPE41; cyan,
PPE15
interaction found between the motifs because the side chain of WxG motif of PPE15
turns away from the PE/PPE binding interface.
The folding patterns are quite similar for both the known structures of PE25/
PPE41 and PE81–99 and PE151–194. Overlapping of both the pairs reveals the shifting
in the helical direction of about 26 –29 and 20 –23 in α-1 and α-2 and α-2 and α-3
of PE8 and PPE15, respectively. These deviations may occur due to the presence of
proline (Pro35 of PE8, Pro71 of PPE15) or glycine (Gly59 of PE8, Gly39 of PPE15).
The α-2 helix of PPE15 that also contains two highly conserved glycine (Gly22 and
Gly33) at the proximal to the kinks and various highly conserved alanine residues
that are present in PE8 and PPE15. These helical bending, deviations, and presence
of kinks on both PE and PPE to get same extent of bending are required for the
specific PE/PPE pair formation and interaction. Both the electrostatic and hydropho-
bic interactions are present in the formation of the complex of PE25/PPE41 and
PE81–99/PPE151–194. Ser48 in the α-2 helix of PE8 makes hydrogen bond (conserved
hydrogen bond) with Tyr154 in the α-5 helix of PPE15. This hydrogen bond
formation is critical for the minimal binding of the protein complex. All the four
helices make several hydrophobic contacts, and at the upper and lower areas of the
complex, distinct salt bridges and hydrogen bonds are present. One salt bridge
(Glu46 of PE8 with Arg14 of PPE15) and three hydrogen bonds (Gln51 of PE8
with Ser93 of PPE15, His73 of PE8 with Tyr45 of PPE15, and Gln70 of PE8 with
Tyr72 of PPE15) are also identified in PE8/PPE15 interactions. In PE25/PPE41, one
salt bridge (Arg24 of PE25 with Glu37 of PPE41) and one hydrogen bond (Glu17 of
PE25 with Thr48 of PPE41) are found at the interface of binding complex, but these
two interactions are not found in PE8/PPE15 in spite that these residues (Ala24 of
PE8 and Glu37 of PPE15 and Gln17 of PE8 and Val48 of PPE15) are present in the
PE8/PPE15 complex. These interactions help in making the complex more stable
and strong. Arg14, Tyr45, Ser93, and Tyr72 residues of PPE15 are essential for the
PE8/PPE15 interaction (Chen et al. 2017). We can say that for them specificity and
binding affinity of PE/PPE complex adopts unique sets of complementary residues.
164 F. Naz et al.
9.7 PE/PPE Proteins and Host Cell Death Pathways: Effectors

as Novel Targets
The PE/PPE family of proteins is commonly associated to the pathogenic mycobac-

terial species. Individual members of PE/PPE proteins have been demonstrated to
involve in the disease process by providing antigenic variation, immune evasion,
immune quorum sensing, and cell death (Choudhary et al. 2003; Deng et al. 2017;
Nair et al. 2009, 2011; Tundup et al. 2008). The pathogenic strains of mycobacteria
employ inhibition of apoptosis/programmed cell death/cell death as a means to
survive within the infected host macrophages. In contrast, it has been shown that
M.tb induces apoptosis for its successful dissemination and propagation (Davis and
Ramakrishnan 2009). Therefore, inhibition of apoptosis by mycobacteria at an early
stage in the infection process provides the pathogen a favorable niche for the
replication and persistence in the host while at later phase in the infection process
when nutrients are scare and bacterial burden is high, mycobacteria use apoptosis for
its dissemination and successful propagation (Gupta and Gollapudi 2006; Haslinger-
Loffler et al. 2005). In this regard, a recent study by Ahmed J et al. demonstrated that
the M.tb multi-functional, intrinsically disordered protein PE/PPE family member
PPE37 C terminal segment translocates to the nucleus and thereby induces caspase
3-dependent apoptosis of the host cells (Ahmad et al. 2018). The co-operonic
PE/PPE proteins PE25/PPE41 which are secreted by the type VII secretion system
of M.tb have been reported to elicit necrosis rather than apoptosis in RAW 264.7
macrophages (Tundup et al. 2014). It has been thought that induction of necrosis by
M.tb is one of the important strategies deployed to enhance its multiplication,
survival, and dissemination (Dobos et al. 2000; Saunders and Britton 2007). How-
ever, inhibition of apoptosis by virulent mycobacteria in infected macrophages is
believed to help the bacteria escape the immune responses that would be generated
when apoptotic bodies harboring bacterial antigens are taken up and presented by
dendritic cells (Behar et al. 2010). The recombinant purified and M. smegmatis
overexpressing PE/PGRS33 have been demonstrated to induce apoptosis in RAW
264.7 macrophages by signaling through TLR2 and the resultant production of
TNF-α (Basu et al. 2007). Additionally, PGRS domain deletion of PE/PGRS33
resulted in its decreased ability to induce apoptosis (Basu et al. 2007). PE/PGRS33
has also been shown to localize in the Jurkat T cells mitochondria following
overexpression to exhibit apoptotic effect (Balaji et al. 2007; Cadieux et al. 2011).
Recombinant M. smegmatis overexpressing PE/PGRS33 demonstrated increased
ability in inducing necrosis and had a better survival in cell and mouse infection
models (Dheenadhayalan et al. 2006). However, wild-type M. smegmatis induces
apoptosis in a PE domain-dependent manner (Cadieux et al. 2011). Therefore, it is
believed that surface-localized PE/PGRS33 is involved in the modulation of host cell
death pathways in several different ways for which the exact mechanism of action
remains to be elucidated.
PE/PGRS18 is a cell envelope-associated protein of M.tb. PE/PGRS18 enhances
the survival of recombinant overexpressing M. smegmatis within the macrophages
possibly through the attenuation of apoptosis. These effects are largely attributed to
the ability of recombinant M. smegmatis to alter the cytokine profile of the infected
macrophages. PE/PGRS18 expressing M. smegmatis leads to the decreased produc-
tion of IL-1β and IL-6 and increased production of IL-12 early in the infection
process, demonstrating their role in physiology and pathogenesis of M.tb (Yang et al.
2017). Another PE/PGRS family member, PE/PGRS41, has been shown to be
associated with the cell envelope of recombinant overexpressing M. smegmatis.
Overexpression of PE/PGRS41 leads to the enhanced survival of recombinant
bacteria within the infected macrophages, and this effect was largely attributed due
to the decreased apoptosis and autophagy, two of the important host innate defense
mechanisms. PE/PGRS41 inhibits the production of pro-inflammatory cytokines
IL-1β, TNF-α, and IL-6. It inhibits the apoptosis by modulating the activation of
caspase 1, caspase 9, and caspase 3, whereas inhibition of autophagy by PE/PGRS41
is mediated through the inhibition of ATG-8. Interestingly, PE/PGRS41 helps in the
dissemination of the overexpressing strain of M. smegmatis by inducing the necrosis
(Deng et al. 2016). An important member of PE/PPE protein family PE13 has been
shown to modulate the host-pathogen interactions through signaling via p38-ERK-
NFκB pathways. Overexpression of PE13 in M. smegmatis demonstrated its cell
wall association. Recombinant expression in M. smegmatis and infection studies
showed that it enhances the survival of recombinant bacteria and also increased its
ability of resistance to different stresses. Overexpressing strain of PE13
demonstrated increased ability to induce the host cell death by increasing apoptosis,
but the underlying mechanism remains to be explored (Li et al. 2016).
PPE32 another member of PE/PPE protein family, which was found to be
associated with the cell wall, modulates the cell death pathway, i.e., apoptosis,
by the activation of caspases 1, 3, and 9. PPE32 induced the apoptosis of
macrophages infected with recombinant PPE32 harboring M. smegmatis, possibly
through the activation of endoplasmic reticulum stress-related pathways and
subsequent activation of Map kinase, MAPK ERK1/2 (Deng et al. 2016).
PE/PPE protein family member PPE44 has been demonstrated to be a virulence
factor; its expression was found higher in the M.tb-infected guinea pig lungs
(Yu et al. 2017). Yu Z et al. showed by heterologous expression of PE44 in
M. smegmatis that it is a cell wall-associated protein. Infection studies with
recombinant bacteria expressing PE44 showed that it leads to the enhanced
secretion of IL-6 and IL-12 through NF-κB-P38-ERK1/2 axis. Infection of
macrophages with recombinant M. smegmatis displayed increased secretion of
LDH. PE44 induces enhanced apoptosis in infected macrophages, thus
demonstrating its involvement in controlling cell death pathways, but the underly-
ing mechanism is not known (Yu et al. 2017). PE11 a PE/PPE family member was
earlier reported to be overexpressed after 24 h of starvation, or transient acid
exposure suggested a role in pathogen persistence or dormancy, particularly its
involvement in fatty acid metabolism (Fisher et al. 2002). Heterologous expression
in M. smegmatis showed its cell wall association. Infection of macrophages with
recombinant bacteria harboring PE11 leads to its enhanced survival. Its
overexpression manipulates IL-6, IL-10, IL-4, and TNF-α secretion and also
induces increased level of necrosis of infected macrophages as demonstrated by
166 F. Naz et al.
increased release of LDH, establishing its role in manipulating cell death (Deng
et al. 2015; Singh et al. 2016). PE9 and PE10 are only of the two clusters that
comprise two PE genes in tandem. It has been recently demonstrated that these two
proteins physically interact with each other and form heterodimer. PE9/PE10
complex interacts with innate immune receptor TLR4 and leads to the enhanced
secretion of IFN-β, IL-10, and IL-1β in treated macrophages. Interaction of
PE9/PE10 to TLR4 elicits increased expression of pro-apoptotic genes bax, bam,
and bid and activation of effector caspase, caspase 3, thus inducing increased
apoptosis in the treated macrophages. This study therefore demonstrated the role
of PE9/PE10 in manipulating the multiple hallmarks of apoptosis and thus impor-
tance of these genes in modulating the M.tb pathogenesis (Tiwari et al. 2015).
The PE/PPE family member, PE/PGRS5, expresses in the later stages of M.tb
infection, i.e., in human granulomas. The PGRS domain of PE/PGRS5 harbors an
intrinsically disordered endoplasmic reticulum-targeting sequence and targeted to
endoplasmic reticulum. It induces TLR4-dependent endoplasmic reticulum stress
response and generation of NO and oxidative stress responses. PGRS domain of
PE/PGRS5 leads to the activation of caspase 8, thereby inducing apoptosis in treated
macrophages. This demonstrates an earlier unknown function of PE/PGRS5 in
endoplasmic reticulum stress-mediated induction of cell death (Grover et al. 2018).
This topic is illustrated in Fig. 9.4.
Fig. 9.4 Diagrammatic representation of role of PE and PPE proteins in cell death. TLR, Toll-like
receptor
9.8 Conclusion
The abundance of PE/PPE protiens in the genome of slow-growing mycobacteria

implies some important functions of this protein family in the life cycle of bacteria.
Although a substantial progress has been made in the biochemical and molecular
characterization since their discovery, their sybilline structural characterestics are
still puzzling researchers. The presence of disordered regions in this family is
additionally perplexing. The disorder regions in these proteins suggest their interac-
tive potential and the ability of moonlighting functions. This higher level of intrinsic
disorder suggests the positive evolutionaty selection. The study of these dynamic
areas will complement the structural studies that are yet scarce in case of these
PE/PPE proteins. The structural studies will aid in target development as well as
biochemical characterization of hypothetical proteins. An extensive effort is required
to reval the atomic coordinates of these proteins with their inhibitors to develop
novel drug targets. Moreover, basic understanding of role of these proteins in
manupilating host cell death pathways could provide insights into novel therapeutic
approaches to curb infection.
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org/10.1111/2049-632X.12096
Comparative In Silico Analyses Reveal
Crucial Factors for Virulence, Antigenicity, 10
and Evolution in M.tb
Yadvir Singh
Abstract
The evolution of pathogenic mycobacteria includes not only loss of nonessential
genes for survival in the host but also horizontal gene transfer-mediated gene
acquisition critical for pathogenicity. Despite this pressure of reductive evolution,
some multigene families have instead expanded down the evolution.
In silico comparative analyses of these multigene families (mce, mmpL, sigma,
TCSS, and PE/PPE) in the context of virulence in M.tb, i.e., phenotypic differ-
ence between H37Rv and H37Ra, have intensified the importance of PE/PPE gene
family.
Although Mycobacterium indicus pranii (MIP), a nonpathogenic soil myco-
bacterium, is positioned much above Mycobacterium tuberculosis in evolutionary
line, it shares 75% of M.tb antigens. Upon comparing MIP with another vaccine
candidate, M. vaccae, in terms of sharing antigens with M.tb, further highlighted
the importance of PE/PPE proteins. This comparative antigenic analysis has also
pointed to the PGRS domain acquisition by the PE proteins and further expansion
through gene duplication events. These results have suggested gene co-option as
another key player in mycobacterial evolution besides horizontal gene transfer
and genomic reduction.
In conclusion, this chapter is an attempt to use in silico methodology to
uncover the importance of PE and PE_PGRS proteins in virulence, antigenicity,
and host immunomodulation in tuberculosis.
Keywords
PE/PPE gene family · Gene co-option · MIP · In silico analyses
Y. Singh (*)
Department Molecular Biology, Max Planck Institute for Infection Biology, Berlin, Germany
e-mail: singh@mpiib-berlin.mpg.de

https://doi.org/10.1007/978-981-32-9413-4_10
172 Y. Singh
Abbreviations
BLAST Basic local alignment search tool

CNP Carbon, nitrogen, and phosphate
ENA European Nucleotide Archive
GRAVY Grand average of hydropathicity
MAC M. avium complex
MCE Mammalian cell entry
MIP Mycobacterium indicus pranii
MMPL Mycobacterial membrane protein large
NCBI National Center for Biotechnology Information
PE Pro-Glu
PGRS Polymorphic GC-rich sequence
PPE Pro-Pro-Glu
RND Resistance, nodulation, and cell division proteins
RR Response regulator
SK Sensor kinase
SNVs Single-nucleotide variations
TCSS Two-component signal transduction systems
10.1 Introduction
10.1.1 Multigene Families in M.tb
In 1905, a parental strain of current laboratory strains H37Rv and H37Ra, H37, was
isolated from a human patient. Later, this strain evolved in two different strains
H37Rv and H37Ra, exhibiting different phenotypes in terms of virulence (Steenken
Jr. and Gardner 1946). H37Rv exhibit a virulent phenotype in contrast to H37Ra,
which exhibits an avirulent phenotype. H37Rv and H37Ra are still used in
laboratories as reference strains around the world to study M.tb.
In 1998, Stewart Cole and colleagues published the first genome sequence of
Mycobacterium tuberculosis (H37Rv, a laboratory strain) (Cole et al. 1998). This
sequence data has provided a novel and significant understanding into the virulence
of M.tb and documented the occurrence of a few highly duplicated gene families. For
competent replication and transcription, huge pressure is always on pathogenic
bacteria like M.tb to preserve their condensed genomes. Gene duplication facilitates
the functional load on a single gene by dispensing it between the duplicate copies.
Further, down the evolution, it also helps to explore new functions through
acquisitions of variations. In M.tb, gene duplication events have contributed toward
the emergence of multigene families encompassing ~ 50% of the proteome (Tekaia
et al. 1999), i.e., mammalian cell entry (mce) gene family, pe/ppe gene family, and
10 Comparative In Silico Analyses Reveal Crucial Factors for Virulence. . . 173
mycobacterial membrane protein large (mmpL) gene family (Mulder et al. 2009).
Expansion of these gene families down the evolution highlights the significance of
their use by the pathogenic bacteria (Sassetti and Rubin 2003). Some of the
multigene families have been linked to pathogenesis of M.tb, i.e., mammalian cell
entry (mce) gene family, mycobacterial membrane protein large (mmpL) gene
family, sigma factor family, and two-component signal transduction systems.
10.1.2 Multigene Families Important in the Pathogenesis

of Tuberculosis
10.1.2.1 Mammalian Cell Entry (mce) Gene Family

Mammalian cell entry (mce) gene family has been shown to play a role in entry and
survival in host macrophages (Arruda et al. 1993). MCE proteins were first
recognized as macrophage cell entry proteins upon expression in nonpathogenic
E. coli (Arruda et al. 1993). Four mce operons are present in M.tb genome (mce1–4).
Each mce operon comprises 8–13 genes. Overall, 36 members (Table 10.1) exclud-
ing Rv0590A (a probable continuation of mce2B) have been described in this family
(Zhang and Xie 2011). The role in transmembrane transportation by MCE proteins is
indicated by the presence of transmembrane helices (Casali and Riley 2007). Fur-
ther, at the N-terminal anchoring in the membrane, MCE proteins also contain a
hydrophobic stretch of amino acid residues (Chitale et al. 2001). These features
correlate their cell surface location with proposed function in host-pathogen
interactions (Harboe et al. 2002). Furthermore, a mutation study in mce operons
had been shown to cause attenuation of M.tb virulence (Gioffre et al. 2005).
10.1.2.2 Mycobacterial Membrane Protein Large (mmpL) Gene Family

Mycobacterial membrane protein large (mmpL) gene family encodes for 13 RND
proteins (Table 10.1) (resistance, nodulation, and cell division proteins) in
mycobacteria and mainly consists of multidrug resistance pumps (Domenech et al.
2005). MMPL7 and MMPL8 proteins have been linked to complex lipid transport
(Cox et al. 1999; Converse et al. 2003). Mutation studies have shown that MmpL4,
MmpL5, MmpL7, MmpL8, MmpL10, and MmpL11 were associated with M.tb
virulence in the mice model (Lamichhane et al. 2005).
10.1.2.3 Sigma Factors Gene Family

Alternative sigma factors control transcription in response to a particular stimulus in
mycobacteria (Bashyam and Hasnain 2004). The ratio of alternative sigma factors to
genome size is highest among the obligate pathogens (Rodrigue et al. 2006). The
sigma factor family permits M. tuberculosis to modulate multiple phases of host-
pathogen interactions like adhesion, entry, intracellular replication, and spreading to
other sites by regulating the temporal expression of specific regulons (Rodrigue et al.
2006). This family has 13 members (Table 10.1), namely, sigma A to sigma
M. SigA, SigC, SigE, SigF, SigH, and SigL proteins were linked to M.tb virulence
(Smith 2003).
174
Table 10.1 List of members of M.tb H37Rv mce gene family, mmpL gene family, sigma factor gene family, TCSS, and PE/PPE (Kohli et al. 2012)
Mammalian cell entry (mce) gene family
Rv0165c Rv0166 Rv0167 Rv0168 Rv0169 Rv0170 Rv0171 Rv0172 Rv0173 Rv0174
Rv0586 Rv0587 Rv0588 Rv0589 Rv0590 Rv0590A Rv0591 Rv0592 Rv0593 Rv0594
Rv1963c Rv1964 Rv1965 Rv1966 Rv1967 Rv1968 Rv1969 Rv1970 Rv1971 Rv3494c
Rv3495c Rv3496c Rv3497c Rv3498c Rv3499c Rv3500c Rv3501c
Mycobacterial membrane protein large (mmpL) gene family
Rv0202c Rv0206c Rv0402c Rv0450c Rv0507 Rv0676c Rv1145 Rv1146 Rv1183 Rv1522c
Rv1557 Rv2339 Rv2942 Rv3823c
Sigma factors gene family
Rv0182c Rv0445c Rv0735 Rv1189 Rv1221 Rv2069 Rv2703 Rv2710 Rv3223c Rv3286c
Rv3328c Rv3414c Rv3911
Two-component signal transduction systems (TCSS)
Rv0260c Rv0490 Rv0491 Rv0600c Rv0601c Rv0602c Rv0757 Rv0758 Rv0818 Rv0844c
Rv0845 Rv0902c Rv0903c Rv0981 Rv0982 Rv1027c Rv1028c Rv1032c Rv1033c Rv1626
Rv2027c Rv2884 Rv3132c Rv3133c Rv3143 Rv3220c Rv3245c Rv3246c Rv3764c Rv3765c
Y. Singh
10
5. PE/PPE gene family

Rv0151c Rv0152c Rv0159c Rv0160c Rv0285 Rv0335c Rv0916c Rv1040c Rv1088 Rv1089
Rv1169c Rv1172c Rv1195 Rv1214c Rv1386 Rv1430 Rv1646 Rv1788 Rv1791 Rv1806
Rv2099c Rv2107 Rv2328 Rv2408 Rv2431c Rv2519 Rv2769c Rv3020c Rv3477 Rv3622c
Rv3650 Rv3746c Rv3872 Rv3893c Rv0109 Rv0124 Rv0278c Rv0279c Rv0297 Rv0532
Rv0578c Rv0742 Rv0746 Rv0747 Rv0754 Rv0832 Rv0833 Rv0834c Rv0872c Rv0977
Rv0978c Rv0980c Rv1067c Rv1068c Rv1087 Rv1091 Rv1243c Rv1325c Rv1396c Rv1441c
Rv1450c Rv1452c Rv1468c Rv1651c Rv1759c Rv1768 Rv1803c Rv1818c Rv1840c Rv1983
Rv2098c Rv2126c Rv2162c Rv2340c Rv2371 Rv2396 Rv2487c Rv2490c Rv2591 Rv2615c
Rv2634c Rv2741 Rv2853 Rv3097c Rv3344c Rv3345c Rv3367 Rv3388 Rv3507 Rv3508
Rv3511 Rv3512 Rv3514 Rv3590c Rv3595c Rv3652 Rv3653 Rv3812 Rv0096 Rv0256c
Rv0280 Rv0286 Rv0304c Rv0305c Rv0354c Rv0355c Rv0388c Rv0442c Rv0453 Rv0755c
Rv0878c Rv0915c Rv1039c Rv1135c Rv1168c Rv1196 Rv1361c Rv1387 Rv1548c Rv1705c
Rv1706c Rv1753c Rv1787 Rv1789 Rv1790 Rv1800 Rv1801 Rv1802 Rv1807 Rv1808
Rv1809 Rv1917c Rv1918c Rv2108 Rv2123 Rv2352c Rv2353c Rv2356c Rv2430c Rv2608
Rv2768c Rv2770c Rv2892c Rv3018c Rv3021c Rv3022c Rv3125c Rv3135 Rv3136 Rv3144c
Rv3159c Rv3343c Rv3347c Rv3350c Rv3425 Rv3426 Rv3429 Rv3478 Rv3532 Rv3533c
Rv3539 Rv3558 Rv3621c Rv3738c Rv3739c Rv3873 Rv3892c
Comparative In Silico Analyses Reveal Crucial Factors for Virulence. . .
175
176 Y. Singh
10.1.2.4 Two-Component Signal Transduction Systems Gene Family

Two-component signal transduction systems (TCSS) consist of a membrane-
localized histidine sensor kinase (SK) involved in recognizing the external signal
and a response regulator localized cytoplasmically (RR). M.tb uses TCSS for
environmental adaptation within the host. TCSSs have also been shown to be
regulating many physiological processes, i.e., competence, sporulation, drug resis-
tance, CNP (carbon, nitrogen, and phosphate) use, and virulence (Krell et al. 2010).
TCSSs consist of 11 core members, 5 orphaned RRs, and 2 orphaned SKs
(Table 10.1), and most of them have been shown to be associated with virulence
(Bretl et al. 2011). Compared to other bacterial species of similar genome size,
M. tuberculosis has a smaller number of complete TCSSs, specifying the evolution
of M.tb as a primarily intracellular human pathogen.
10.1.2.5 PE/PPE Gene Family

The PE/PPE gene family consists of proteins with conserved signature motif Pro-Glu
(PE) and Pro-Pro-Glu (PPE) at the N-terminus, respectively. This gene family
consists of 107 PE and 69 PPE members (Table 10.1). PE gene family has
been further branched into pe and pe_pgrs gene family. In PE_PGRS, PE motif is
followed by polymorphic GC-rich repetitive sequences (PGRS) at the C-terminal
domain, which code for glycine and alanine repeats (Brennan and Delogu 2002;
Cole et al. 1998). These glycine and alanine repeats further show variations in
number, size, and sequence between different PE_PGRS proteins (Brennan and
Delogu 2002).
10.1.3 Pathogenic M.tb vs Nonpathogenic Mycobacteria
The mycobacterium genus comprises more than 120 species, which can be easily
grouped into non-pathogens like M. smegmatis opportunistic pathogens like
M. avium and true pathogenic bacteria like Mycobacterium tuberculosis (M.tb).
The genome size of nonpathogenic mycobacteria is larger than that of true pathogens
suggesting that gene loss is a significant part of continuing evolution of slow-
growing pathogenic mycobacteria (Brosch et al. 2002; Ahmed et al. 2008). There
is also an acquisition of genes specific to intracellular survival in the host by M.tb.
Comparative proteomic analysis of M.tb with nonpathogenic predecessor
mycobacteria like Mycobacterium indicus pranii (MIP) has helped to elucidate the
reasons of pathogenicity of M.tb (Singh et al. 2014).
10.1.3.1 Mycobacterium Indicus Pranii (MIP)

MIP is a saprophytic, nonpathogenic mycobacterium and taxonomically positioned
in M. avium complex (MAC) of mycobacteria (opportunistic pathogens) (Ahmed
et al. 2007; Saini et al. 2009). MIP has been used as an immunomodulator in various
diseases (Ahmad et al. 2011; Katoch et al. 2008; Rakshit et al. 2012; Talwar 1999),
i.e., an effective commercial immunotherapeutic vaccine “Immuvac” against leprosy
(Nath 1998). Further, animal trials of MIP have shown that it can impart a protective
response against M.tb infection and is under testing in phase III human clinical trials
(Gupta et al. 2012; Gupta et al. 2009; Saini et al. 2012). This broad-spectrum
antigenic potential of MIP is justified by an exceptional taxonomical position of
MIP which leads to sharing more B- and T-cell antigens with other pathogenic
mycobacteria including M.tb and M. leprae (Saini et al. 2012; Singh et al. 1992;
Yadava et al. 1991).
10.1.4 Gene co-Option: Another Key Player in M.tb Evolution
Gene co-option includes modification of existing function or acquisition of a new

function of a gene, possibly via gene duplication. In absence of gene duplication,
genes can still gain a new function either by a change in coding sequences or gain of
novel domains. While in presence of gene duplication, co-option can be acquired by
either subfunctionalization (partial conservation of function) of the paralogous genes
or neo-functionalization (gain of novel function) (True and Carroll 2002). Gene
co-option has also been shown to elucidate the presence of virulence factors among
environmental and pathogenic Rhodococci bacteria, which belong to the same
taxonomical order like M.tb (Letek et al. 2010). Gene co-option could elucidate
the emergence of pathogenic bacteria from nonpathogenic bacteria through adaptive
changes in the niche factors.
10.2 Methods Used in In Silico Comparative Proteomics of M.tb
The first step for a comparative analysis is collection of gene sequences from various
databases. Gene database of National Center for Biotechnology Information (www.
ncbi.nlm.nih.gov/gene) and European Nucleotide Archive sequence database (www.
ebi.ac.uk/ena) are valuable sources for listing and FASTA sequences of genes. With
the help of these databases, FASTA sequences of these genes are searched in ENA
database for corresponding genes in another strain/species after considering nucleo-
tide position number (base range) as a reference point. Furthermore, BLASTn
(nucleotide BLAST) tool from NCBI is helpful to explore differences between two
strains/species in terms of insertions, deletions, and single-nucleotide variations
(SNVs). Pipeline for in silico analyses is shown in Fig. 10.1.
Through various in silico analyses, the genetic and proteomic differences among
these established gene families have been linked to the context of different virulence
phenotypes of H37Rv and H37Ra. Various parameters can be calculated and com-
pared using in silico tools. With the help of T-coffee align program, many proteins
across these multigene families have been found to contain substitutions, insertions,
and deletions in amino acid sequence between H37Rv and H37Ra. ProtParam tool
from ExPASy portal (http://www.web.expasy.org/protparam/) is able to calculate
various physicochemical properties like instability index, aliphatic index, and grand
average of hydropathicity (GRAVY) which have been further explained in the
following paragraphs.
178 Y. Singh
Fig. 10.1 Pipeline for in silico analyses for comparative proteomics of M.tb
The instability index correlates with the stability of the protein in vitro. Instability
index >40 suggests unstable protein and < 40 suggests stable protein. Some
dipeptides are present in different frequency in unstable vs stable proteins. The
composition of such dipeptides can be correlated to the protein stability (Guruprasad
et al. 1990).
The aliphatic index of a protein denotes the relative volume enveloped by
aliphatic side chains. Increase in temperature can increase aliphatic hydrophobicity;
therefore it has a positive correlation with the thermal stability of globular proteins
(Ikai 1980).
Protein solubility is indicated by GRAVY, where a negative value correlates with
hydrophilicity and positive with hydrophobicity. Hydrophilicity will increase the
hydrogen bonding with water molecules and which in turn will increase solubility.
ProtParam shows GRAVY score using Kyte-Doolittle’s hydropathic indices of the
amino acids (Kyte and Doolittle 1982).
GlobPlot (http://www.globplot.embl.de) tool indicates globularity in the proteins.
GlobPlot analyzes the disordered and globular regions of a protein, using the
Russell/Linding propensities (Linding et al. 2003). Globular domains in protein
molecules might dispense special functions. Therefore, loss or gain of function in a

protein can also be correlated to addition or deletion of globular domains.
Phosphorylation of amino acid residues like serine and threonine can play
important role in protein-protein interactions. NetPhosBac (http://www.cbs.dtu.dk/
services/NetPhosBac-1.0/) predicts serine and threonine phosphorylation sites in
proteins using neural network algorithms on the dataset of bacterial proteins (Miller
et al. 2009).
Besides physicochemical properties, antigenicity can also be calculated in silico,
i.e., VaxiJen. Through VaxiJen v2 server (http://www.ddg-pharmfac.net/vaxijen/
VaxiJen/VaxiJen.html), antigenicity index of proteins can be measured with an
accuracy of 70–89%. This tool utilizes an alignment-free, autocross-covariance-
based method to calculate the antigenicity of a protein sequence (Doytchinova and
Flower 2007).
10.3 Results
10.3.1 Outcome of Comparative Proteomics of M.tb Strains
Nucleotide and amino acids sequence comparison of multigene families across the
two strains of M.tb revealed the occurrence of a few differences between H37Rv and
H37Ra genome in the perspective of established gene families except PE/PPE for
virulence. Our in silico analyses (Kohli et al. 2012) revealed that substitutions only
were not very substantial but insertions/deletions along with substitutions led to
remarkable dissimilarities in the characteristics of these proteins.
MCE multigene family has a wider distribution among nonpathogenic and
pathogenic mycobacterium; therefore mce operons in M.tb may not be directly
involved in imparting virulence to M.tb (Haile et al. 2002). Interestingly our in silico
analysis (Kohli et al. 2012) did not reveal any differences that could be associated
with the attenuation of the H37Ra strain. The results were similar in MmpL gene
family except for one member. The mmpl13 gene of H37Rv and H37Ra was a case of
gene splitting into mmpL13a and mmpL13b and another study predicted them to be
functional open reading frames based on an another in silico analysis (Sandhu and
Akhter 2015). A CDS mismatch in mmpl13b gene was detected which led to
globular domain extension and phosphorylation site gain in H37Ra (Kohli et al.
2012). The significance of this observation is further subjected to the lack of any
designated function of mmpl13b gene in virulence or pathogenesis of mycobacteria.
Further in the same analyses, 1 out of 13 sigma factors, sigM, exhibited nucleotide
and amino acid sequence change between H37Rv and H37Ra. sigM (Rv3911) had a
one nucleotide insertion causing a frameshift and subsequent truncation of the
protein. This resulted in globular domain extension and phosphorylation site loss
in H37Ra. Another study has shown that sigM gene might be important for long-term
in vivo adaptation (Agarwal et al. 2007).
In case of TCSS, a single-nucleotide change only in the case of phoP (response
regulator of phoP-phoR TCSS) was observed, which led to an amino acid
180 Y. Singh
substitution from serine to leucine and subsequent phosphorylation site loss in

H37Ra (Kohli et al. 2012). This substitution has been shown to decrease
DNA-binding capacity of protein and further associated with H37Ra attenuation
phenotype, although complementation partly restored the H37Ra persistence in a
murine macrophage infection model (Lee et al. 2008).
10.3.2 Comparative Proteomics Highlights the Importance

of PE/PPE Multigene Family in Virulence
PE/PPE gene family, which is the largest multigene family in M.tb, has also
exhibited a number of differences in its member proteins among H37Ra and H37Rv
than any other multigene family (Kohli et al. 2012). Another study regarding
comparative genome analysis of H37Rv versus H37Ra has shown that pe/ppe
multigene family exhibited the most variations between H37Rv and H37Ra
(Zheng et al. 2008). Besides phoP, variation in PE/PPE proteins might be the most
important factor involved in attenuation of H37Ra. Despite a large number of
proteins in this family, only a few have been characterized so far (Akhter et al.
2012). The study of these proteins is very challenging as only one PE/PPE protein
pair has been crystallized so far (Strong et al. 2006). The absence of proteolytic sites
in these proteins further makes proteomic studies such as mass spectroscopy
extremely difficult (Abdallah et al. 2009).
Nucleotide sequence comparison and protein annotation have revealed that some
proteins were absent in H37Ra, i.e., PE10, PE_PGRS49, PE_PGRS56, and
PE_PGRS60. Interestingly, some of the PE/PPE genes that have been linked to the
pathogenicity of H37Rv also exhibited clear amino acid sequence differences with
H37Ra in this study (Kohli et al. 2012).
For example, wag22 protein in H37Rv (Rv1759c) has been shown to be expressed
during infection (Espitia et al. 1999). In a previous study, inactivation of wag22
homologue in Mycobacterium marinum was linked to defective replication in
macrophages and subsequent diminished survival in granulomas (Ramakrishnan
et al. 2000). In H37Rv, this protein exhibited fibronectin-binding properties and
antigenicity at C-terminal end (Espitia et al. 1999); while in H37Ra, a truncation at
C-terminal end (MRA_1772) resulted in aliphatic index change, globular domain
expansion, and loss of phosphorylation sites. The PGRS domain has been shown to
protect the PE_PGRS protein from ubiquitin-proteasome-dependent (UPD) proteol-
ysis (Koh et al. 2009). The significance of the PGRS domain in the PE_PGRS
protein stability has been previously shown by using PE_PGRS fused to green
fluorescent protein (Brennan and Delogu 2002). PE_PGRS22 and wag22 have
exhibited C-terminal truncation, which might have resulted in altered in vivo stabil-
ity in H37Ra (Kohli et al. 2012). In the same study, a single-nucleotide deletion in
PE_PGRS59 (Rv3595c) homologue in H37Ra has resulted in the formation of two
PE_PGRS proteins, namely, MRA_3634 and MRA_3635 in H37Ra. MRA_3635
and Rv3595c are both stable and share the same globular domain position.
Furthermore, both MRA_3635 and MRA_3634 are more hydrophobic and contain
additional phosphorylation sites compared to Rv3595c.
Rv3508 (PE_PGRS54) has been shown to play a role in nitric oxide stress
response and hypoxia during infection (Yu et al. 2011). Our in silico analyses
have revealed substitutions and indels in the amino acid sequence of Rv3508
homologue in H37Ra. Furthermore, loss of phosphorylation sites in H37Ra might
render this protein unresponsive to such stress responses leading to an attenuated
phenotype.
PPE18 has been shown to be expressed on the surface and therefore interacts with
TLR2 receptor on macrophages. PPE alters the host innate immune balance by
repressing IL-12 production and promoting IL-10 production to help bacteria to
persist in the host (Nair et al. 2009). Differential interaction of PPE18 with PE13 and
PE31 has been shown to modulate host-pathogen interactions (Mukhopadhyay and
Balaji 2011). In H37Ra, PE13 and PPE18 expression was suppressed in macrophages
(Li et al. 2010).
In silico analysis has shown differences in amino acid sequence of PPE18 protein
between H37Rv and H37Ra (Kohli et al. 2012). Specific indels led to alterations in
serine/threonine phosphorylation sites in H37Rv and H37Ra, which might further
cause altered protein-protein interactions. GlobPlot analysis has revealed extension
of N-terminal globular domain of H37Rv PE13 homologue in H37Ra, which might
led to altered function. Similarly, N-terminal extension in MRA_1801 (Rv1787
homologue) caused formation of new serine/threonine phosphorylation sites, leading
to alterations in signaling and host–pathogen interactions. Strains of M. avium
lacking MAV_2928 (homologue of Rv1787) have been linked to compromised
virulence by preventing vacuole acidification and phagosome-lysosome fusion
(Jha et al. 2010). PPE31 (Rv1807) has been shown to be important for in vivo
growth during infection in mice (Sassetti and Rubin 2003). Interestingly, in silico
analysis showed this protein to be more stable in H37Ra than H37Rv with obvious
physiological implications.
These in silico analyses have revealed differences in many functionally unknown
PE/PPE proteins, i.e., PE24, PPE5, PPE9, PPE13, PE_PGRS22, PE_PGRS25,
PE_PGRS36, PE_PGRS40, and PE_PGRS47. PPE5 homologue MRA_0313 has
N-terminal extension leading to an acquisition of an additional globular domain
which might impart an additional role to the protein. Similarly in PE_PGRS40
(Rv2371) homologue MRA_2394, N-terminal extension made the protein globular
less hydrophobic in H37Ra. Likewise in PPE54 (Rv3343c) homologue MRA_3384,
deletion of amino acid stretch in the central region resulted in a globular domain loss,
probably causing a loss of function in H37Ra. Indels and substitutions in Rv2098c
homologue MRA_2113 also caused change in globular domains of the protein in
H37Ra. In Rv1091 homologue MRA_1102, acquisition of a globular domain
resulting from deletions in C-terminal end was observed.
182 Y. Singh
Fig. 10.2 Comparative

antigenicity index analysis
between M.tb, MIP, and
M. vaccae (Singh et al. 2014)
10.3.3 Outcome of Comparative Antigenicity Index Analysis
3166 proteins of M.tb (H37Rv) exhibited antigenicity index >0.4 and were listed as
putative antigens. 152 out of ~ 170 PE/PPE multigene family members displayed
antigenicity index >0.4. Further, some of them lied in the high-end range of
antigenicity index (> 1) and therefore emphasized their importance in terms of
antigenicity in M.tb (H37Rv). For a meaningful interpretation, another rapidly
growing soil mycobacterium Mycobacterium vaccae was also included. Mycobacte-
rium vaccae has also been explored as a vaccine candidate against TB in some
clinical trials (Yang et al. 2010; Yang et al. 2011). 2498 of the putative M.tb antigens
exhibited amino acid sequence similarity with MIP proteins (Fig. 10.2) than
M. vaccae which had 2209 putative antigen homologues.
10.3.4 Outcome of Gene Duplication in PE/PPE Family
152 PE/PPE antigens with >0.4 antigenicity index (29 PE proteins, 62 PE_PGRS,
and 61 PPE proteins) were compared with MIP homologues. Out of the 62 PE_PGRS
proteins, 52 showed amino acid sequence similarity with MIP_06644 (Singh et al.
2014). MIP_06644 exhibited antigenicity index value of 0.4428 (antigenic) and
instability index value of 56.07 (unstable).
After MIP_06644, MIP_03868 exhibited sequence similarity with these
52 proteins. Seven MIP PE proteins and 21 MIP PPE proteins exhibited amino
acid sequence similarity with 26 PE proteins and 58 PPE proteins of M.tb (H37Rv),
respectively. These results have highlighted the prevalence of duplication in
PE_PGRS gene family. Along with duplication, increased antigenicity might be
crucial for M.tb in modulating the host immune response for its persistence.
10.3.5 Comparative Genomic Profiling and Its Prospect in Infectious

Diseases Like TB
Comparison of mycobacterial strains or species at genomic, transcriptional, and

proteomic level is highly helpful to understand the evolution of differences in
various traits like virulence phenotype, growth rate characteristics, host specificity,
etc. Pan genomic analysis further broadens the approach by comparing many species
together at the same time. At the genus level, pan mycobacterial genomic analysis
has revealed that slow growers have lost growth enhancer genes, i.e., LivFGMH
operon, shaACDEFG, and MspA porin (Wee et al. 2017). Such analyses can further
address phenotypic differences among M.tb strains, between M.tb complex, between
M.tb and NTM (nontuberculous mycobacteria) (Behr 2014).
In vivo or in vitro comparative transcriptional and proteomic analyses are very
essential to validate the in silico findings of genomic analysis. For example, M. bovis
and M.tb exhibit more than 99% nucleotide sequence similarity and yet have distinct
host preferences. Recently one study with an in-depth transcriptional and transla-
tional profiling of M. bovis and M.tb in bovine macrophages has revealed differential
gene expression between these two species (Malone et al. 2018). Similar studies are
also needed to understand the precise functional role of PE/PPE multigene family in
mycobacterial evolution.
10.4 Conclusion
With the help of comparative genomics studies, it is evident that Mycobacterium

tuberculosis (M.tb) has evolved by gene loss (Ahmed et al. 2008), as well as gene
acquisition responsible for pathogenicity and virulence through horizontal gene
transfer (Veyrier et al. 2011). Despite reductive evolution, certain gene families in
M.tb have expanded in size, possibly through various gene duplication events, i.e.,
mce, pe/ppe, and mmpL gene family. Gene duplication events have impacted nearly
50% of M.tb proteome evolution as many multigene families (Tekaia et al. 1999).
Our in silico analysis (Kohli et al. 2012) has revealed that well-studied multigene
families in the context of M.tb virulence, i.e., mce, mmpL, sigma, and TCSS, might
not be adequate to describe the phenotypic difference between H37Rv and H37Ra.
This demands more investigation of relatively unexplored PE/PPE multigene family
as this family exhibited many nucleotide sequence variations upon a comparison
between H37Rv and H37Ra. Mycobacterium indicus pranii (MIP) is positioned much
above Mycobacterium tuberculosis in an evolutionary timeline and is a nonpatho-
genic soil mycobacterium (Saini et al. 2009); it has shared 75% of antigens (both
putative and validated) of M.tb in the in silico analysis (Singh et al. 2014). This study
further revealed that MIP shared not only a larger number of M.tb antigens but also
contains larger repertoire of highly antigenic PE/PPE proteins compared to another
vaccine candidate M. vaccae. Therefore compared to M. vaccae, MIP might be better
in exhibiting antigenic variation and wider antigenic exposure to employ protective
immunity against M.tb. This comparative antigenic analysis clearly implies gene
184 Y. Singh
co-option as another key player in mycobacterial evolution besides gene loss and
gene acquisition (horizontal gene transfer). Supporting this notion, an interesting
aspect of gene duplication event was observed in highly antigenic PE_PGRS gene
family. Our analyses (Singh et al. 2014) revealed PE_PGRS gene family expansion
through gene duplication, with improved stability and antigenicity, as a valuable
catalyst in the host immune response modulation by M.tb. In conclusion, these in
silico analyses have attempted to show that PE/PPE multigene family is crucial for
virulence, antigenicity, and host-pathogen interactions in tuberculosis. Furthermore,
PE/PPE multigene family might also be important for an antituberculosis vaccine
efficacy and prominent gene duplication events have highlighted gene co-option as a
key player in M.tb evolution. This is the first study which has shown gene co-option
as another key player in mycobacterial evolution besides gene loss and gene
acquisition. Therefore, a systemic functional analysis of proteins from this multigene
family will be highly helpful in devising effective preventive (vaccines) and drug
therapies against tuberculosis.
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Importance of Cell Wall-Associated
Poly-a-L-Glutamine in the Biology 11
of Pathogenic Mycobacteria
Rajni Garg, Rajesh Mani, Manish Gupta, Deeksha Tripathi,

Harish Chandra, Rakesh Bhatnagar, and Nirupama Banerjee
Abstract
Mycobacterium tuberculosis (Mtb), the formidable scourge known to mankind
since ancient times, has remained untamed despite vigorous scientific research in
the field. In the last several decades, significant advances have been made to study
this pathogen; however, a lot more remains in the realm of unknown. The
complex and unique cell wall of the bacterium is a major factor contributing to
the unrestrained success of the pathogen in infecting millions around the world.
Since the discovery of this bacterium, numerous studies have attempted to
unravel the complexities of mycobacterial cell envelop to characterize individual
constituents and their importance in pathobiology of Mtb. Major components of
the cell envelop of mycobacteria such as lipid-linked polysaccharides-
R. Garg
Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore, Karnataka, India
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, Karnataka,
India
R. Mani · M. Gupta · N. Banerjee (*)
Molecular Biology and Genetic Engineering Laboratory, School of Biotechnology, Jawaharlal
Nehru University, New Delhi, Delhi, India
D. Tripathi
Department of Microbiology, School of Life Sciences, Central University of Rajasthan, Ajmer,
Rajasthan, India
H. Chandra
Department of Microbiology, School of Biomedical & Pharmaceutical Sciences, Babasaheb
Bhimrao Ambedkar University, Lucknow, India
R. Bhatnagar
Banaras Hindu University, Varanasi, Uttar Pradesh, India

https://doi.org/10.1007/978-981-32-9413-4_11
190 R. Garg et al.
lipoarabinomannan (LAM), dimycolyl trehalose (cord factor), sulfolipids, and

mycolyl-arabinogalactan-peptidoglycan (mAGP) complex have been
investigated extensively. However, a lesser known molecule, poly-α-L-gluta-
mine/glutamate (PLG), that constitutes ~10% of dry weight of cell wall has not
attracted as much attention. As early as 1990, Hirschfield et al. isolated PLG as
insoluble material and showed its association with the Mtb cell wall. In the last
few years, our group has been working to identify enzymes that may play a role in
the synthesis/assembly and localization of this polymer in the cell wall of
mycobacteria. Our recently published work has shown that PLG by itself is
weakly immunogenic in mice, but when combined with protein antigens, it can
stimulate different arms of the T helper-mediated responses, demonstrating its
potential to act as an adjuvant (Mani et al. 2018).
Keywords
Poly-α- L-glutamine/glutamate (PLG) · Mycobacterial virulence · Glutamine
synthetase · Adjuvant
Abbreviations
GS glutamine synthetase
LAM lipoarabinomannan
mAGP mycolyl-arabinogalactan-peptidoglycan
Mtb Mycobacterium tuberculosis
PGS polyglutamate synthase
PLG poly-α- L-glutamine/glutamate
TB tuberculosis
XDR extensively drug resistant
11.1 Introduction
Tuberculosis (TB) continues to be one of the major causes of morbidity and

mortality across the globe (Pai et al. 2016). One of the key reasons for unrelenting
success of Mycobacterium tuberculosis (Mtb), the causative agent of TB, is its
distinct cell wall. The latter is an elaborate structure distinct from other eubacteria
(Brennan 2003). It is highly impervious in nature, acting as a protective barrier
against hydrophilic drugs (Jankute et al. 2015). The emergence of multidrug-
resistant (MDR) and extensively drug-resistant (XDR) bacteria has necessitated
the need for studying different mycobacterial cell wall components to develop
effective new-generation antitubercular therapeutics that can target them individu-
ally. Poly-α-L-glutamine/glutamate (PLG) is one such cell wall component found
11 Importance of Cell Wall-Associated Poly-a-L-Glutamine in the Biology. . . 191
only in pathogenic mycobacteria. In this chapter, we have attempted to review the

earlier research related to PLG and present the work done in our laboratory in the
context of biology of mycobacteria. Here, we discuss some of the yet unexplored
aspects of the glutamine polymer as revealed by our recent research.
11.2 Molecular Structure of Mycobacterial Cell Wall
Mycobacteria possess a cell wall that is markedly distinct from other eubacteria.
Mycobacterial cell wall has a complex architecture with an inner and an outer layer
(Brennan 2003). The inner layer forming the core of the cell wall contains unique
mycolyl-arabinogalactan-peptidoglycan (mAGP) complex that surrounds the cyto-
plasm (Brennan 2003), with the peptidoglycan facing the plasma membrane, while
mycolic acids are present at the distal end. In the outer lamella, proteins are present
along with glycolipids and some long- and short-chain fatty acids (Brennan 2003;
Alderwick et al. 2015).
11.2.1 The Inner Lamella
The peptidoglycan (PG) in the inner lamella of the mycobacterial cell wall is unique
in several ways. It contains N-acetyl glucosamine (NAG) linked to N-modified
muramic acid in a β (1–4) configuration (Alderwick et al. 2015). Some modifications
in muramic acid are acetyl in nature as in other eubacteria, while others are glycolyl
in nature (Mahapatra et al. 2000, 2005; Alderwick et al. 2015). Secondly, the
mycobacterial PG is an extensively cross-linked (70–75%) structure. Tetrapeptide
side chains consisting of L-alanyl-D-iso-glutaminyl-meso-diaminopimelyl-D-alanyl-
D-alanine are cross-linked with peptides of neighboring glycan chains by a 3–3 bond
in addition to the regular 3–4 bond (Wietzerbin-Falszpan et al. 1970; Alderwick et al.
2015). Thirdly, D-glutamate and diaminopimelic acid (DAP) are amidated and lastly
some of the D-glutamate or L-alanine residues are modified with glycine (Kotani et al.
1970; Draper 1971; Draper et al. 1987). The arabinogalactan (AG) is a
heteropolysaccharide which is covalently attached to 10–12% of muramic acid
residues of the PG via a phosphodiester linkage (Amar and Vilkas 1973).
11.2.2 The Outer Lamella
Outer lamella of the wall has soluble proteins and lipids, which primarily function as
the signaling molecules of mycobacteria (Brennan 2003). The phthiocerol-
containing lipids such as phthiocerol dimycocerosate, glycolipids like
lipoarabinomannan (LAM), lipomannan, dimycolyl trehalose (cord factor),
sulfolipids, and the phosphatidylinositol mannosides are the typical constituents of
the mycobacterial cell wall (Brennan 2003).
192 R. Garg et al.
In the last few years, substantial work on Mtb cell wall has strengthened our
understanding of biogenesis and structure of mAGP complex (Meroueh et al. 2006;
Berg et al. 2007). Based on experimental evidences, it was reported that poly-α-L-
glutamine (PLG) peptides are non-covalently associated with the peptidoglycan in
virulent mycobacteria (Hirschfield et al. 1990). In this chapter, we have described
our research elucidating the biochemical and immunological properties of this
unique constituent of the mycobacterial cell wall.
11.3 Poly-a-L-Glutamine (PLG)
Though extensive work has been done on the AGP complex and other cell wall
components, the poly-α-L-glutamine (PLG) polymer has received little attention
despite its discovery several decades ago (Wietzerbin-Falszpan et al. 1973; Juana
Wietzerbin et al. 1975; Phiet et al. 1976). In the last few years, our group has focused
on decoding the biological significance of PLG.
In 1976, Phiet et al. analyzed cell walls of various strains of mycobacteria, e.g.,
M. tuberculosis (Mtb) W115, five strains of M. bovis BCG Pasteur, Glaxo, Phipps,
Tice, and Montreal for their PLG content. This study revealed that the amount of
PLG in the cell wall positively correlated with degree of virulence of the strain and,
secondly, PLG was absent in the avirulent M. bovis BCG Montreal strain (Phiet et al.
1976). In an attempt to unveil molecules responsible for immunoreactivity of Mtb
cell wall, Hirschfield et al. isolated the insoluble polymer PLG along with a 23 kDa
immunogenic protein by trifluoromethanesulfonic acid treatment of the insoluble
mAGP complex (Hirschfield et al. 1990). The unique physical properties of the
polymer, particularly its insolubility in detergents, organic acids and bases,
chaotropes, and many common solvents, resulted in its co-purification with the
cell walls. Isolation of the polymer by nondegradative means suggested its
non-covalent association with the cell wall. PLG constituted 10% of the mycobacte-
rial cell wall weight (Hirschfield et al. 1990). It was also suggested that PLG might
serve as a nitrogen and carbon reserve for the bacterium to survive in adverse
conditions (Hirschfield et al. 1990).
11.4 Biogenesis of PLG
11.4.1 Role of Glutamine Synthase
Till date there is no direct evidence showing the specific role of GS (glutamine
synthetase) in PLG synthesis; however various reports have suggested a close link
between the two. Several studies have shown that amount of PLG in the cell wall is
dependent on the expression and activity of glutamine synthetase (GS encoded by
glnA1 gene), secreted in copious amount by virulent mycobacteria (Harth et al. 1994;
Harth and Horwitz 1999; Tullius and Horwitz 2003; Chandra et al. 2010). Harth
et al. in 1994 successfully purified the GS enzyme encoded by glnA1 gene from Mtb
to near homogeneity and demonstrated its catalytic activity in the synthesis of
glutamine. GS is a 680 kDa dodecameric protein. Since this enzyme is extracellular
in nature, it was proposed that it provides glutamine for the assembly of PLG layer
(Harth et al. 1994).
Harth and Horwitz investigated the effect of GS inhibitors on the amount of PLG
and growth of pathogenic and nonpathogenic mycobacteria. Addition of inhibitor
MSO (L-methionine-S-sulfoximine) rapidly inhibited GS activity of Mtb under
in vitro conditions (Harth and Horwitz 1999). Moreover, when added to the cultures
of pathogenic mycobacteria, it inhibited extracellular GS in a concentration-
dependent manner, but had nearly no effect on cellular GS, probably because of
its inability to cross the mycobacterial cell wall. Treatment of Mtb with MSO
reduced PLG content also in the cell wall, indicating the involvement of GS in
PLG synthesis (Harth and Horwitz 1999). The inhibitor selectively reduced growth
of all pathogenic mycobacteria, which release GS extracellularly, but did not affect
nonpathogenic mycobacteria or other bacteria, which do not secrete GS.
Our group for the first time generated an M. bovis glnA1 mutant and established
direct link between GS activity and PLG formation (Chandra et al. 2010). The
mutant strain showed marked reduction in the PLG content, concomitant with
decrease in the cell wall strength. The cell wall of the mutant became highly sensitive
to a variety of physical and chemical stresses, namely, sonication, SDS, and lyso-
zyme, respectively. Disruption of the glnA1 gene resulted in attenuation of the
mutant in the THP-1 cells and BALB/c mice (Chandra et al. 2010). The mutant
showed reduced biofilm formation and increased sensitivity to antitubercular drugs
such as rifampicin and D-cycloserine. Further, expression of the mycobacterial glnA1
in M. smegmatis led to synthesis and export of GS in the extracellular medium and
concomitant formation of PLG in the cell wall (Chandra et al. 2010). The above
results showed involvement of glnA1 in the synthesis of PLG in virulent
mycobacteria.
11.4.2 Role of Rv0574c (Polyglutamate Synthase-like Enzyme)
Formation of PLG in the cell wall as a corollary to glutamine synthetase activity

provided potential targets for therapeutic intervention against mycobacterial infec-
tion. However, high level of similarity between the catalytic domains of the myco-
bacterial GS and the human homologue called for identification of alternate enzymes
involved in PLG biosynthesis. We approached the problem by mining mycobacterial
genome for homologues of genes shown to encode enzymes catalyzing synthesis/
localization of polyglutamate polymer in the cell wall of other bacteria. We identified
Rv0574c locus annotated as polyglutamate synthase (PGS)-like enzyme showing
95% identity with other capsule synthesis proteins, e.g., CapA from Streptomyces
194 R. Garg et al.
violaceusniger Tu 4113, and 25.8% identity with CapA, the polyglutamate synthase
of B. anthracis (Garg et al. 2015). Based on the above search, we hypothesized that
the encoded protein might play a role in the formation of PLG in the cell wall of
mycobacteria. The expression pattern of Rv0574c (PGS) through different growth
phases correlated with the amount of PLG in Mtb cell wall. The expression levels of
the protein peaked in late log phase with concomitant accumulation of PLG which
continued to increase till the stationary phase (Garg et al. 2015). The Rv0574c gene
is reported to be a member of the dormancy regulon, a 48-gene cluster controlled by
the transcriptional regulator DevR (Dev R-Dev S two-component system) in
mycobacteria (Park et al. 2003; Sousa et al. 2007; Garg et al. 2015). We observed
that signals such as hypoxia, nitric oxide, and carbon dioxide reported to activate
DevR-mediated dormancy regulon, upregulated transcription of Rv0574c gene also
(Garg et al. 2015). In the light of the above observations, it is proposed that as the
cells progress into stationary phase, the above host signals trigger the dormancy
regulon (Park et al. 2003; Sousa et al. 2007) and result in PGS-dependent increase in
PLG contents in the cell wall to help the bacterium adapt/resist the host-generated
stressful environment.
To find the precise role of Rv0574c in mycobacterial biology, a gene deletion
mutant was created. The mutant strain contained less PLG in the cell wall than the
wild-type strain, though the reduction was not as high as the glnA1 phenotype (Garg
et al. 2015). Whether decrease in the amount of extractable PLG was due to lack of
synthesis or localization is a subject of future studies. Like the glnA1 mutant, the
Rv0574c mutant also exhibited increased sensitivity to lysozyme and SDS, which
could be due to lower PLG content in the cell wall. The mutant was defective in
biofilm formation compared to the parent Mtb strain, which produced biofilm rich in
PLG, implying its involvement in maintaining cellular integrity and architecture of
the cell wall (Garg et al. 2015).
The importance of Rv0574c in the survival of the bacterium was further
highlighted by the loss in virulence of the mutant strain, which was attenuated for
growth in the macrophages. Replication of the ΔRv0574c strain was highly reduced
(1 log10) in the lungs of BALB/c mice after 8 weeks of infection (Garg et al. 2015).
The mutant showed minimal histopathological changes in the lung parenchyma of
the infected mice, indicating substantial reduction in pathogenicity (Garg et al.
2015). PLG content in the cell wall of the mutant was also reduced, though not as
severely as the glnA1 phenotype. A small but statistically significant increase in PLG
content was seen when Mtb PGS was overexpressed in M. smegmatis, suggesting an
accessory function of this protein in PLG synthesis/transport. Thus our results
suggest that PGS is important for protecting the bacterium against the deleterious
effects of host defense mechanisms like NO, CO, hypoxia, etc. and also makes the
cell wall resistant to chemical and mechanical stresses with the help of PLG,
facilitating its entry into the dormant stage. Further studies are underway to decipher
the catalytic properties of this protein in the PLG synthesis.
11.5 Regulation of PLG Synthesis
Nitrogen metabolism plays a critical role in bacterial physiology. In the cell,

synthesis of nitrogenous biomolecules depends mainly on two nitrogen donors,
glutamate and glutamine (Mehta et al. 2004; Newsholme et al. 2003). In
mycobacteria, GS enzyme is responsible for assimilation of inorganic nitrogen to
glutamine (Umbarger 1978). Since glutamine synthesis is a high energy consuming
process, the glnA1 gene encoding GS is under stringent control, both at transcrip-
tional and posttranslational levels. It has been reported earlier that the glnA1 gene in
Mtb has two promoter sequences P1 and P2 in the upstream regulatory region (Harth
and Horwitz 1997). The transcript length varied when the bacteria were grown in
low and high nitrogen condition, suggesting that transcription initiates from two
different sites depending on the availability of nitrogen (Harth and Horwitz 1997).
Furthermore, when the nitrogen concentration is low, the GlnR protein acts as a
positive regulator by binding to the P1 promoter and activates transcription (Tiffert
et al. 2008). While at higher nitrogen concentrations, the GS enzyme is adenylylated
by GlnE protein, rendering it inactive to avoid depletion of the cellular glutamate
levels by conversion to glutamine (Harper et al. 2008). In the light of these
observations, we were motivated to examine the involvement of individual
promoters of the glnA1 gene in regulating PLG synthesis at different nitrogen
concentrations in mycobacteria.
For our studies, we used M. smegmatis host which produces a nonsecretory and
yet functional GS enzyme. Secondly, it does not contain PLG in the cell wall under
normal growth conditions (Harth et al. 1994; Harth and Horwitz 1997). For this
purpose, we generated several recombinant strains MSFP, MSP1, and MSP2
(M. smegmatis expressing MtbglnA1 with dual promoter sequence and with individ-
ual P1 or P2 promoters, respectively) (Tripathi et al. 2013). The recombinant strains
produced extracellular GS enzyme depending on the concentration of nitrogen in the
growth medium. Effect on PLG formation was also examined in response to nitrogen
availability by isolating and estimating PLG during growth. It was revealed that at
low nitrogen concentrations, the P1 promoter was primarily responsible for the
synthesis of major amount of the extracellular glutamine synthetase, while the P2
promoter had no apparent role in the production of the enzyme. At high nitrogen
condition also, the amounts of both intra- and extracellular GS enzyme produced by
the MSP2 strain were lower than the MSP1 strain. Further, the synthesis of PLG
directly correlated with the magnitude of extracellular glutamine synthetase activity,
implying that it is the P1 promoter that mainly drives the synthesis of both GS and
PLG under low nitrogen condition only. Our results with separate individual
promoters corroborate earlier studies (Mehta et al. 2004) that high nitrogen concen-
tration acts as a negative regulator of glutamine synthetase in mycobacteria. Sec-
ondly, it appears that for P2 promoter, nitrogen may not be the real trigger for
activation in the context of glutamine synthetase or PLG synthesis. It would be
interesting to examine if there are other signals or factors involved in regulation of
the P2 promoter in mycobacteria. The synthesis of PLG during low nitrogen
availability and its absence in the cell wall in nitrogen sufficiency indicated that
196 R. Garg et al.
Fig. 11.1 Regulation of glutamine synthetase (glnA1) gene in response to nitrogen availabil-
ity. In nitrogen abundance condition, GlnE inhibits activity of GS, leading to reduced glutamine
synthesis and thus PLG. In nitrogen limitation condition, GlnR positively regulates GS and leads to
formation of glutamine and thus PLG. Discontinuous arrows and ??? indicate steps in PLG
synthesis and localization that are still under investigation. (Adapted and modified from Tripathi
et al. 2013)
PLG might be a nitrogen storage mechanism in virulent mycobacteria. Figure 11.1

shows pictorial representation of transcriptional and posttranslational regulation of
glnA1 and hence PLG, in response to nitrogen availability.
11.6 Immunostimulatory Properties of PLG
The components of Mtb cell wall are known to interact with a variety of host factors
upon infection and hence play a crucial role in the modulation of host immune
responses (Karakousis et al. 2004). The strong immune-stimulatory properties of the
cell wall components present in complete Freund’s adjuvant (CFA), an emulsion of
mannide monooleate and heat-killed Mtb, are attributed to muramyl dipeptide
(MDP) and trehalose 6,60 -dimycolate (TDM) (Kasmar et al. 2014). Unfortunately,
due to multiple toxic effects of CFA, e.g., inflammation and pain at the site of
injection, abscess, intraperitoneal granulomas, abdominal adhesions, and lymphoid
hyperplasia in the mice, it cannot be used for humans, restricting its use to laboratory
studies only (Petrovsky and Aguilar 2004). The adjuvants play a crucial role in
enhancing/modulating the immunogenicity of a vaccine candidate (Reed et al.
2013). The “adjuvant-like” properties of synthetic glutamine-rich self-assembling
peptides and their derivatives were shown to increase IgG1, IgG2, IgG3, and IgM
antibody levels equivalent to that of CFA (Rudra et al. 2010). Taking a cue from
these studies, we investigated the adjuvant potential of PLG, using a variety of
different model antigens.
Our study involved testing of PLG in combination with various prototypical
antigens like the Mtb-secreted antigenic target protein 6 (ESAT-6) and antigen
85B (Ag85B), protective antigen (PA) of Bacillus anthracis and BP26 of Brucella
abortus in the mouse model. The results revealed that no specific antibodies were
produced against PLG after repeated immunizations with the peptides alone,
indicating its weak immunogenicity in mice. In contrast, adjuvantation of the
antigens with PLG triggered a strong antigen-specific immune response compared
to antigen alone. A significant increase in the serum IgG levels and its isotypes
(IgG1, IgG2a, and IgG2b), especially IgG2a, against all the above antigens occurred
by adjuvantation with PLG, suggesting induction of a Th1-biased immune response.
Parallel to the humoral response, a robust production of Th1-specific IFN-γ, TNF-α,
and IL-2 and Th2-specific IL-6 and IL-10 cytokines was also observed in the
cultured splenocytes from PLG adjuvanted mice as a memory response to recall
antigen. Higher IgG production and isotype switching to IgG2a in the PLG
adjuvanted mice group is consistent with enhanced production of Th1-stimulatory
IFN-γ and TNF-α cytokines. A simultaneous induction of antigen-specific IgG and
its isotype and cellular memory in PLG adjuvanted mice groups indicated stimula-
tion of both Th1 and Th2 effector arms of immunity.
Recently, Th17 cells have emerged as a major immune mediator against TB due
to secretion of high levels of IL-17A. The latter results in increased chemokine
production, T- and B-cell localization, and migration of Mtb-infected alveolar
macrophages in the lung granulomas, enabling containment of Mtb in the lungs
(Van Dis et al. 2018). Earlier, a vaccine formulation containing H1 (a fusion protein
of Ag85B and ESAT-6) and H28 (a fusion protein of Rv2660, Ag85B and TB10.4)
antigens with CAF01 adjuvant was shown to elicit long-lasting Th17 responses in
mice against Mtb infection (Lindenstrøm et al. 2012). Notably, adjuvantation of
ESAT-6 with PLG in mice elicited significantly high antigen-specific IL-17 produc-
tion compared to antigen alone or dimethyldioctadecylammonium/monophosphoryl
lipid-A (DDA/MPL, a common Th1 inducer) adjuvanted group, suggesting a strong
Th17 response. Thus the ability of PLG to induce a broad, multifaceted immune
response makes it a potential candidate adjuvant molecule for exploration, particu-
larly for chronic infectious diseases caused by intracellular pathogens like Mtb and
human immunodeficiency virus (HIV), where Th1-type immune response is neces-
sary for effective clearance and protection (Burton and Moore 1998; Simon et al.
2011; Ottenhoff and Kaufmann 2012).
198 R. Garg et al.
The efficacy of ESAT-6 subunit vaccine candidate was tested after Mtb challenge
in mice. Addition of PLG in the ESAT-6 vaccine formulation increased the secretion
of Th1-specific cytokines significantly, showing development of immune memory,
marked with concomitant reduction of bacterial burden in the lung and spleen of the
C57BL/6J mice. Further, the protective efficacy of the formulation was also reflected
on the survival of mice. The median survival time of the mice immunized with
antigen alone was 66 days compared to 205 days of PLG adjuvanted mice, which
was close to 224 days observed with BCG vaccination (Mani et al. 2018). In the
above study ESAT-6, a weak immunogen was chosen as the vaccine candidate to
allow PLG to manifest its immunomodulatory activity optimally, without being
masked by a strong antigen. It would be interesting to evaluate long-term effect on
survival/protection with stronger immunogens like Ag85B or CFP10 or a combina-
tion of multiple proteins vis-à-vis the BCG vaccine.
The molecular mechanism underlying the action of PLG as a vaccine component
remains unclear. Some of the bacterial products are immune-stimulatory by virtue of
their similarity with microbial structures, referred to as pathogen-associated molec-
ular patterns (PAMPs), and recognized as foreign element by the host (Reed et al.
2009; O’Hagan et al. 2017). Indeed, it is known that microbial derivatives mediate
their action through pattern recognition receptors (PRRs) such as Toll-like receptors
or TLRs on the surface of antigen-presenting cells (APCs) mainly macrophages and
dendritic cells (Dowling and Mansell 2016). The interaction of PAMPs in a subunit
vaccine with the PRRs on the surface of APCs activates the downstream signaling
pathways, releasing proinflammatory cytokines along with the expression of MHC
class I and II molecules to elicit the adaptive immune response (Liechtenstein et al.
2012). It is likely that PLG may act as a PRR ligand on different APC subsets and
stimulate the Th1, Th2, and Th17 pathways of the immune response. Alternatively,
PLG may transport antigen proteins by entrapping or by non-covalent association,
facilitating their entry into the APCs, thereby augmenting the antigen-specific
immune responses. Such a mechanism of adjuvantation has been reported earlier
for synthetic poly(lactic-co-glycolic) acid (PLGA) particles (Manish et al. 2013),
virus-like particles (Gao et al. 2018), and immune-stimulating complexes also
(Sanders et al. 2005).
Our investigations exploring the biological properties of PLG are still in the
preliminary stages. The physicochemical properties of PLG, an insoluble, homopol-
ymer of L-glutamine, seem to be largely responsible for the immunomodulatory
action of this mycobacterial cell wall component (Phiet et al. 1976; Hirschfield et al.
1990; Mani et al. 2018). In view of its consistent immune-stimulatory profile across
protein antigens of diverse bacterial origin, our research has laid the foundation for
developing PLG as an efficient Th1-specific adjuvant. However, an essential prereq-
uisite for the development of any biological product is the assessment of its toxico-
logical properties. We found the PLG preparation nontoxic to THP1 macrophages
and in C57BL/6J mice, yet comprehensive safety evaluation of the product is
imperative in diverse animal models. Secondly, studying the mechanistic orchestra-
tion of host immune response and pharmacokinetics of PLG in different hosts will
pave the way to exploit its full potential for developing a more precise pathogen/
vaccine-specific immunomodulator molecule. This study demonstrated that the PLG

peptides of Mtb cell wall act like an archetypical adjuvant by stimulating both the
humoral and cell-mediated immune response of the host.
In the past couple of decades, extensive research has unraveled the biochemical
nature of mycobacterial cell wall, advancing our knowledge regarding individual
components of the envelope. However, for some reason poly-α-L-glutamine did not
attract as much attention as the others. It was our endeavor to take the existing
knowledge about poly-α-L-glutamine forward and explore its significance in the
biology of mycobacteria. Our efforts have suggested some interesting aspects of this
cell wall constituent preparing the ground for future studies.
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32:525–532
Cellular Stress Responses
and Immunological Regulations During 12
Mycobacterium tuberculosis Infection
Nooruddin Khan, Gillipsie Minhas, K. Kala jyothi, and Jyoti Sharma
Abstract
Tuberculosis (TB) is one of the most devastating infectious diseases caused by
Mycobacterium tuberculosis (MTB). A high percentage of mortality and morbid-
ity associated with TB has been reported globally with the highest number of
cases reported in Asia. MTB is known to enter a state of dormancy and pheno-
typic drug resistance, when it is exposed to multiple stress conditions in the host
microenvironment, and thereby it survives asymptomatically in latent phase in
the host for decades and even for a lifetime. This raises the need for improved
vaccine, drugs, and therapeutics, which could be achieved by a better understand-
ing of the host-microbe interactions as well as immune responses during the
infection. Recent studies have highlighted the importance of host cellular stress
response pathways, such as unfolded protein response (UPR), oxidative stress
response, integrated stress response (ISR), and autophagy during various
infections. However, the role of these host stress response pathways during an
MTB infection in the modulation of the immune response against the microbe is
poorly understood. Therefore, through this chapter, we will highlight the cellular
stress response pathways and various molecular mechanisms through which
MTB influences the host innate as well as the adaptive immune response during
infection, which might aid toward better design and development of therapeutics
and vaccine candidates against TB.
Keywords
Mycobacterium tuberculosis · Tuberculosis · Immune response · Stress response ·
ER stress
N. Khan (*) · G. Minhas · K. Kala jyothi · J. Sharma

Department of Biotechnology and Bioinformatics, School of Life Sciences,
University of Hyderabad, Hyderabad, Telangana, India
e-mail: noor@uohyd.ac.in

https://doi.org/10.1007/978-981-32-9413-4_12
204 N. Khan et al.
Abbreviations
APC antigen-presenting cells

ATF activating transcription factor
BCG bacille Calmette-Guerin
BiP binding immunoglobulin protein
CHOP CCAAT-enhancer-binding protein homologous protein
CLR C-type lectin receptor
CR complement receptors
DALYs disability-adjusted life years
DC dendritic cells
eIF2 eukaryotic initiation factor 2
eNOS endothelial nitric oxide synthase
ER endoplasmic reticulum
ESAT6 early secretory antigenic target 6 kDa
IFN interferon
IL interleukin
iNOS induced nitric oxide synthase
IRE1 inositol-requiring enzyme 1
ISR integrated stress response
JNK c-Jun N-terminal kinases
LC3 microtubule-associated protein 1A/1B-light chain 3
LPS lipopolysaccharide
MAPK mitogen-activated protein kinases
MDR multidrug resistance
MHC major histocompatibility complex
mTOR mammalian target of rapamycin or mechanistic target of rapamycin
NK cells natural killer cells
NLR NOD-like receptors
NO nitric oxide
NOD nucleotide oligomerization domain
NOS nitric oxide synthase
nNOS neuronal nitric oxide synthase
PAMPs pathogen-associated molecular patterns
PERK protein kinase RNA-like ER kinase
PI3P phosphatidylinositol 3-phosphate (PI3P)
PKR protein kinase R
PRR pattern recognition receptors
RIPE rifampicin, isoniazid, pyrazinamide, and ethambutol
RLR RIG-I-like receptors
ROS reactive oxygen species
SPA surfactant protein A
STING stimulator of IFN genes
12 Host Response During MTB Infection 205
TB tuberculosis
TBK1 TANK-binding kinase 1
TLRs Toll-like receptors
TNF tumor necrosis factor
UPR unfolded protein response
XBP1 X-box-binding protein 1
12.1 Introduction
Tuberculosis (TB) poses a major health threat in developing nations despite the
available therapies. It is one of the top three infectious and fatal diseases after AIDS
and malaria. Over one-third of the population is infected with the pathogen world-
wide, and it is estimated that over a 100 million people have died of the disease in the
past 100 years, while about 35 million are predicted to die between the years 2000
and 2020 (Zumla et al. 2015). With 9.6 million new cases of TB per year being
reported worldwide, it becomes even more indispensable to contain the pathogen. In
addition, the disease burden of TB way surpasses mortality and contributes to a high
level of disability-adjusted life years (DALYs) lost due to illness and outdoes
malaria in this aspect (WHO 2016). According to WHO report 2016, 60% of
recorded TB cases have been contributed majorly by six developing countries:
India, Indonesia, China, Nigeria, Pakistan, and South Africa (WHO 2016).
TB is caused by bacillus Mycobacterium tuberculosis (MTB), a notorious
pathogen that has become difficult to eradicate with BCG which is the only vaccine
available even after a century of its discovery (Luca and Mihaescu 2013). It is
acknowledged that only 5% of individuals infected with MTB develop active
disease, while the remaining 95% contain the pathogen in an asymptomatic
inactive form (dormant/latent form), which is resistant to the current anti-
mycobacterial drugs used for treating active disease. Streptomycin was the first
effective drug used against active TB. Currently, a combination of rifampicin,
isoniazid, pyrazinamide, and ethambutol (RIPE therapy) for 2 months followed by
rifampicin and isoniazid for another 4 months is the treatment of choice for
uncomplicated active infection (Joshi 2011). However, emergence of multidrug-
resistant (MDR) strains has made it difficult to target the pathogen with available
drug therapies. Many second-generation recombinant BCG vaccines are in differ-
ent stages of clinical trials for their immunogenicity to exterminate both forms of
the disease (Kaufmann et al. 2010).
TB pathogenesis, in general, is a delicate interplay between the mechanisms
employed by the host to evade the pathogen and the strategies employed by the
pathogen to survive within the unfavorable host environment. During an MTB
infection, the host tries to maintain homeostasis by eliciting various forms of cellular
fate mechanisms like necroptosis, apoptosis, and autophagy, among which apoptosis
and autophagy have been documented as one of the crucial innate defense
206 N. Khan et al.
mechanisms (Behar et al. 2010, 2011; Bradfute et al. 2013; Du et al. 2013). The
pathogen also employs various rescue strategies like induction of anti-inflammatory
cytokines and subsequent decrease in TNF-α-mediated apoptosis, upregulation of
specific microRNAs that attenuate autophagy to escape the host immune system. A
better understanding of the host and pathogen defense mechanisms has therefore
become even more important to target the pathogen and to develop new
interventions to circumvent the disease.
12.2 Mycobacterium tuberculosis Pathogen and Infection
TB is an ancient disease that has been linked with the evolution of human race with
reports dating as old as 5000 years. MTB is one of the oldest described bacterial
pathogens and is thought to have evolved about 3 million years ago (Gutierrez et al.
2005). With time, various breakthroughs in the field of microbiology have been
made, such as the development of BCG vaccine (Calmette and Boquet 1921) and the
discovery of drugs like streptomycin, isoniazid, rifampicin, etc. (Van Scoy and
Wilkowske 1999). All these developments have led to a boost in treatment and TB
research. However, the calamity of TB has reemerged with the advent of different
multidrug-resistant strains of MTB.
The pathogen was first described by Robert Koch in 1882, as a nonmotile,
non-sporulating, slow-growing pathogenic species. It is facultative, intracellular
bacteria that multiply in phagocytic cells like macrophages. The progress of infec-
tion depends on the initial interaction between the pathogen and the host. MTB
infection commences with inhalation of the pathogen, which then travels to alveoli in
the lungs. The bacteria first interact with the resident alveolar macrophages and this
step is an absolute prerequisite for MTB to establish an infection in a susceptible host
lung. Though the primary role of macrophages is protective, intake of the pathogen
by these defense cells is vital for the establishment of infection. The interaction
prompts a primary immune response through pro-inflammatory molecules and
recruitment of leukocytes. This early encounter in the lungs itself decides the fate
of the pathogen, whether the bacteria will remain contained in the macrophages as a
latent form or will lead to an active disease. Infection further activates different
mechanisms such as fusion of phagolysosomes, respiratory burst, and production of
reactive oxygen species (ROS) (Giacomini et al. 2001). The response that is initiated
upon binding of the bacteria with the macrophage depends on the type of phagocytic
receptor (Ernst 1998). The most common receptors on the macrophages are termed
as pattern recognition receptors (PRR), which recognize different bacterial
components, termed as pathogen-associated molecular patterns (PAMPs); the com-
plement receptors (C receptors), which are important for bacterial opsonization;
mannose receptors, which assist macrophages in phagocytosis of unopsonized
bacteria; surfactant protein A (SPA), which modulates the activity of other receptors
to enhance macrophage binding and MTB uptake; and scavenger receptors, which
act at later stages of infection to further stimulate macrophages and inflammatory
responses. The binding of PAMPs to the specific receptors initiates phagocytic
pathways, for example, binding to FcR produces reactive oxygen species (ROS) and
phagolysosomal fusion, whereas, in the case of CR3 receptor, the binding inhibits
the respiratory burst (Armstrong and Hart 1975; Kang et al. 2005; Mortaz et al. 2015;
Pahari et al. 2017).
Once internalized, MTB stays in the phagosome and blocks its fusion with the
lysosome. The bacteria act by eliminating phosphatidylinositol 3-phosphate (PI3P)
from the infected cells, which is used in trafficking of lysosome to phagosome and
thus impedes the fusion (Vergne et al. 2005). MTB infection also inhibits the
production of phosphatidylinositol 3-phosphate (PI3P), which is essential for induc-
tion of autophagy (Vergne and Deretic 2010). The cell wall components of MTB,
especially particular lipids, also have an impact as these interact with the cell
membrane of phagosomes and affect their maturation and hence its elimination
(Axelrod et al. 2008).
12.3 Host Defense Mechanisms: Innate and Adaptive Immune

Responses
Each cell has its own defense mechanisms that limit the cellular damage and
ultimately eliminate the pathogen. Most of these responses against a pathogen
invasion is to maintain the cellular homeostasis by decreasing the cellular functions
such as protein translation or inducing cellular processes like apoptosis and
autophagy. A successful infection is dependent on the initial interaction between
the host and the pathogen. In case of MTB, initial exposure to the pathogen occurs
through airborne droplets, which are inhaled and then transported to lungs, where it
is taken up by the resident macrophages through phagocytosis for elimination. The
initiation of the innate arm of the immune system is through the PRRs, which
recognize conserved PAMPs unique to each pathogen and are essential for their
survival and pathogenesis. The PRRs are expressed in almost all immune and
nonimmune cells and majorly on antigen-presenting cells like macrophages and
dendritic cells. They are able to recognize both extracellular and intracellular
pathogens as they are expressed both on the cell surface as well as within the
endosomes and are involved in activating pro-inflammatory signaling pathways
and stimulating phagocytic responses. Based on the function and location, the
PRRs have been classified into four families, Toll-like receptors (TLRs),
nucleotide-binding oligomerization domain-like receptors (NODs), C-type lectin
receptors (CLRs), and RIG-1-like receptors (RLRs) (Takeuchi and Akira 2010).
TLRs are the imperative players with respect to immunity against infectious
agents as they play a critical role in initiating an innate and adaptive immune
response against vast majority of pathogens. They were first identified in Drosophila
and later found to be associated with mammals as a response to bacterial infections
(Leulier and Lemaitre 2008). TLRs are known to act individually or in sets to
recognize molecular signatures of pathogens such as the cell wall components in
response to infection. Host response against MTB has been postulated to involve
TLR signaling as the pathogen is laden with well-characterized TLR ligands that are
208 N. Khan et al.
potent stimuli to activate the Th1-mediated immune response (Jones et al. 2001;
Thoma-Uszynski et al. 2001; Abel et al. 2002; Feng et al. 2003; Basu et al. 2012).
Much work done by researchers has shown that majorly TLR-2, TLR-4, and TLR-9
play phenomenal roles in the induction of immunity against MTB infection (Bafica
et al. 2005; Bhatt and Salgame 2007; Mukherjee et al. 2016). TLR-2 participates in
recognizing the cell wall components of MTB and promoting the macrophages to
secrete TNF-α, IL-12, as well as nitric oxide. Mice deficient in TLR-2, when infected
with MTB, were not able to elicit such responses (Reiling et al. 2002; Drennan et al.
2004). The GC-rich genome of MTB is a very good ligand for TLR-9, which
interacts with CpG motifs that may regulate the progression of granuloma formation
(Ito et al. 2007). The role of TLRs in eliciting immunity against TB is understood
from the work of Velez et al., where the authors showed that polymorphisms in
TLR-2 and TLR-9 are associated with increased susceptibility to TB in different
populations (Velez et al. 2010). The role of TLR-4 however is not well understood.
Single missense mutation in TLR-4 rendered mice infected with MTB is inefficient
in controlling bacterial loads and initiate pro-inflammatory responses, while TLR-4-
deficient mice had no such effect (Jo et al. 2007).
Recently, NOD-like receptors (NLRs) have also shown to play a role in immunity
against MTB. NLRs contain leucine-rich repeats and, unlike TLRs, recognize
intracellular PAMPs in the cytosol. NOD-2 has been shown to play a crucial role
in TLR activation through the production of inflammatory cytokines, TNF-α and
IL-1β, in human macrophages during TB infection (Brooks et al. 2011). Moreover,
NOD-2-deficient mice showed poor innate and adaptive immunity and increased
bacterial loads upon MTB infection (Divangahi et al. 2008).
The early response subsequent to phagocytosis involves activation of innate
immune and Th1 pathways, which leads to granuloma formation (Salgame 2005).
The formation of the granuloma is a protective host response against MTB that can
inhibit the spread of bacteria, thus keeping the infection in latent phase. The
granuloma consists of infected cells and immune cells (differentiated myeloid cells
surrounded by lymphocytes) along with the macrophages and MTB, which is
attacked by CD4+ cells and B cells that restrict the bacteria in the capsule-like
structure (Peters and Ernst 2003).
Alveolar macrophages facilitate both innate and adaptive immune responses to
combat MTB infection. Also, phagocytosis of MTB causes secretion of cytokines
that are pro-inflammatory in nature, such as IFN-γ, TNF-α, IL1-β, IL-6, and IL-12
(Brightbill et al. 1999). Th1 cytokine such as IFN-γ is secreted majorly by the CD4+ T
cells, which plays a central role as host defense in anti-mycobacterial immunity, such
as induction of NOS2 expression and upregulation of MHC class II molecules, which
in turn play important roles in antigen processing and presentation (Harding and
Boom 2010). IFN-γ thus contributes to MTB control by connecting innate and
adaptive immune responses. It has also been shown that IFN-γ enhances the
autophagic control of MTB, while the Th2 cytokines like IL-4 have the opposite
effects (Harris et al. 2007). In addition, the pro-inflammatory cytokine TNF-α acts
synergistically along with IFN-γ during antimicrobial responses against MTB by

playing an important role in activation of phagosome maturation and hence the
development of granuloma to restrict MTB (Garcia et al. 1997). Further, the role of
TNF-α in anti-mycobacterial immunity is evident in case of patients treated with anti-
TNF-α antigens in a variety of inflammatory/autoimmune diseases including TB
(Harris and Keane 2010). TNF-α can induce apoptosis accompanied by the produc-
tion of reactive oxygen intermediates/reactive nitrogen intermediates to destroy the
engulfed bacteria (Rojas et al. 1999; Ciaramella et al. 2002). Actively
involved immune cells such as neutrophils and natural killer (NK) cells, secrete
cytokines, such as IFN-γ, which further stimulates secretion of IL-12 by the
macrophages (Schierloh et al. 2007). These studies emphasize the role of cytokines
in providing protection against various pathogens including MTB. Figure 12.1 shows
the innate immune response elicited upon MTB infection.
Development of adaptive immunity needs close interaction between T cells and
APCs. Upon macrophage activation via antigen presentation through MHC class II
complex, the APCs take up the infected apoptotic macrophages and extracellular
MTB to stimulate CD8+ T cells by cross-presenting MTB antigens via MHC class I
complex. Thus, the CD4+ and CD8+ T cells activate each other to eliminate MTB.
Studies have also highlighted the role of CD8+ cells in infection through cytokine
production, such as IFN-γ and TNF-α, which activate the bactericidal mechanisms.
Another T-cell subpopulation that has also been associated with an early response
toward TB in granuloma formation is the γδT cells (Griffin et al. 1991; Collins and
Kaufmann 2001). Figure 12.2 highlights the adaptive immune response produced
during MTB infection.
Additionally, costimulatory molecules such as B7.1 (CD80) and B7.2 (CD86)
expressed on macrophages and dendritic cells and cytokines produced by these cells
stimulate the T cells and thus facilitate the elimination of MTB. MTB, therefore,
circumvents the action of these inhibitory components by attenuating the expression
of costimulatory molecules or by dampening the production of pro-inflammatory
cytokines.
Another primary host defense response is the release of nitric oxide (NO), a
gaseous free radical molecule produced by a two-step enzymatic reaction catalyzed
by nitric oxide synthases (NOSs). Several free radical studies have revealed that the
production of reactive oxygen and nitrogen species is an innate host defense
mechanism against various types of pathogens including bacteria, viruses, parasites,
and fungi (Nathan and Shiloh 2000; Bermudez 1993). Depending upon the types of
cells they are expressed in, NOSs are of three types: endothelial cells (eNOS),
neuronal cells (nNOS), and as an inducible form (iNOS). While the first two kinds
are constitutively expressed, iNOS is expressed in various types of immune cells and
produces excessive NO during infection and inflammation (Moncada et al. 1991;
Nathan and Xie 1994; Zaki et al. 2005) or upon induction by the inflammatory
cytokines TNF-α, IFN-γ, and IL-1β (Lee et al. 1993). NO and related reactive
nitrogen intermediates have been shown to exhibit anti-mycobacterial activities in
murine models of TB infection. However, the effect of NO on mycobacterial
210 N. Khan et al.
Fig. 12.1 Host innate immune defense mechanisms against Mycobacterium tuberculosis
The interaction of MTB pathogen with macrophages in lungs increases ROS and RNS production
and leads to phagolysosomal fusion. MTB phagocytosis also releases cytokines, IL1β, TNFα, and
IFNγ, which activate different immune responses and granuloma formation to restrict the infection
survival in human macrophages has remained controversial (Rich et al. 1997). Study
has shown NO production in human monocyte-derived macrophages upon infection
with Mycobacterium avium; however, no NO production was demonstrated upon
MTB infection in human monocytes (Denis 1991; Weinberg et al. 1995). Study by
Rich et al. showed that the infected alveolar macrophages are capable of NO
production that can directly or indirectly act as an MTB inhibitor and further impede
intracellular MTB growth in alveolar macrophages (Rich et al. 1997). All these
studies signify a contributory role of NO in controlling mycobacterial infections in
murine macrophages; further investigations are required to ascertain the role of NO
in human macrophages.
Fig. 12.2 Adaptive immune response against Mycobacterium tuberculosis

The host cells upon sensing bacteria trigger series of signaling cascades, which leads to the
activation of innate immune response and release of various innate effector molecules including
cytokines. These cytokines together with other innate molecules further dictate and shape the
effector commitment of the T-cell immune responses
12.4 Host Defense Mechanisms: Stress Response Pathways
Under normal physiology, the body has its own survival mechanisms to maintain the
homeostasis. However, various intracellular or extracellular stress conditions, such
as endoplasmic reticulum (ER), hypoxia or nutrient starvation, can activate a cascade
of stress response signaling events, in the form of unfolded protein response (UPR),
integrated stress response (ISR), oxidative stress response, or autophagy (Fulda et al.
2010; Rubartelli et al. 2013). Eukaryotic cells have various sensors that sense stress
stimuli, such as nutrient deprivation, endoplasmic reticulum (ER) stress, double-
stranded RNA, and heme deprivation. The integration of these stress response
212 N. Khan et al.
pathways with immune regulation has been explored under various inflammatory
and metabolic diseases (Ganguly et al. 2016; Battu et al. 2017), which could be
investigated further for viral and bacterial infections. Viral proteins are well known
to induce stress response due to their dependence for survival on host machinery for
protein synthesis. On a similar note, even bacteria and/or their components can
induce cellular stress in the host that can evoke different reactions (Celli and Tsolis
2015).
ER not only plays a crucial role in folding and modification of new proteins but
also senses different cellular stress (Zhang and Kaufman 2008). Calcium imbalance,
hypoxia, or reactive oxidative stress (ROS) in ER are a few instances that can also
activate the stress response cascade to return to homeostasis. The cascade initiates
through three means: by decreasing the proteins that enter the ER, by elevating the
chaperone levels to regulate the protein folding, and by removing misfolded proteins
by degradation (Kaufman 2002; Kaufman et al. 2002). These pathways also initiate
an immune response. Eventually, if the homeostasis is not retained through these, the
ultimate response is apoptosis. Bacterial infection in the body is also known to
activate the stress sensors in the ER (Pillich et al. 2016). The stress response mainly
increases the phosphorylation of eIF2α that halts the host protein translation and
activates the pro-apoptotic response. The ER stress thus regulates many secretory
and cellular proteins which ultimately can cause accumulation of misfolded proteins,
activating the unfolded protein response (UPR) signaling (Berridge 2002).
MTB infection increases the level of markers indicating toward ER stress. There
are primarily three stress sensors in ER: inositol-requiring enzyme 1 (IRE1α),
protein kinase RNA-like ER kinase (PERK), and activating transcription factor
6 (ATF6). These three proteins act through a chaperone protein, Ig heavy-chain-
binding protein (BiP). Under normal condition, BiP binds to the stress sensors on the
luminal side in ER and keeps them in an inactive state. As the unfolded proteins
accumulate in ER, BiP is released from the stress sensor proteins and binds to the
unfolded proteins. The released sensors act to induce UPR cascade. IRE1 in the
unbound form autophosphorylates and splices an intron from mRNA of X-box-
binding protein 1 (XBP1); the spliced XBP1 acts as a transcription factor for UPR
target genes. Prolonged exposure to ER stress then causes UPR to activate apoptosis
signaling through caspases and C/EBP homologous protein (CHOP) (Zhang and
Kaufman 2008). On a similar note, PERK activates the kinase domain and
phosphorylates the serine residue of eIF2α that inhibits the protein translation and
decreases protein load on ER.
Lim et al. investigated the ER stress mechanism in MTB-induced infection in
mouse macrophages. The authors found the association with ER stress-induced
apoptosis, which confers through caspase 12, present in ER cytoplasm. In response,
it was observed at 24 h of MTB infection that the pathogen resists the cessation of
host protein translation by decreasing the phosphorylation of eIF2α, which helps in
the survival (Lim et al. 2011). The study also found elevated levels of caspase 12 and
caspase 9, which is its precursor postinfection. All three stress sensor proteins were
found to be activated after the infection (Lim et al. 2011).
There are bacterial components that can also induce UPR signaling, such as
lipopolysaccharides (LPS), that is present in the outer membrane of gram-negative
bacteria. Similarly, toxins that are released can also activate UPR. The apoptosis of
infected macrophages releases antigen of mycobacterial origin, early secretory
antigenic target 6 (ESAT6), which can induce ER stress. Choi et al. investigated
the ER stress response in the ESAT6-stimulated A549 human epithelial cells. It was
noted that the presence of ESAT6 elevated the levels of intracellular calcium, which
can cause ROS and ultimately ER stress-induced apoptosis (Choi et al. 2010).
ESAT6 is the virulence factor for MTB that is required for pathogenicity. This
makes ESAT6 a promising candidate for vaccine development (Wang et al. 2009).
ESAT6 treatment also increases phosphorylation of eIF2α and leads to elevated
levels of ATF4 and GADD34 and induces apoptosis through the ASK1/JNK
pathway. It also acts through cleavage of caspase 12 and 4 into their active forms.
Therefore, all these investigations convey that the ER stress triggers apoptosis in
infected macrophages through the PERK pathway which involves phosphorylation
of eIF2α and activation of CHOP.
Another stress response sensor, dsRNA, activated serine/threonine Protein
Kinase R (PKR) is well known to regulate cytokines during viral infections. In
case of viral infections, PKR has a well-established role in the phosphorylation of
eIF2α and thus halting translation. The large levels of IFN-γ, which are induced
upon viral infection, trigger the PKR activity, thus increasing its capacity to recog-
nize viral origin dsRNA. Upon binding to dsRNA, PKR autophosphorylates to an
active protein kinase, which phosphorylates eIF2α (De Haro et al. 1996). The role of
this kinase has also been investigated in MTB infection. Cheung et al. in their study
examined the interaction of primary human blood monocytes with BCG. BCG was
shown to stimulate expression of cytokines such as TNF-α, IL-6, and IL-10 in
monocytes. The authors used PKR inhibitors to demonstrate the involvement of
PKR phosphorylation and activation in the stimulation of these cytokines by BCG.
PKR activation is known to trigger translocation of NFκβ to the nucleus, through
MAPK activation, which ultimately controls the transcription of cytokine genes
(Cheung et al. 2005). BCG treatment did not show any effect on the mRNA levels
of PKR but had an immediate consequence of its kinase activity. Therefore, this
study associates PKR activity with MTB infection, apart from its already known role
in viral infections (D’Acquisto and Ghosh 2001; Williams 2001). Further
investigations are needed to elicit the mechanism behind the PKR-based anti-
mycobacterial activity.
Deprivation of energy, nutrients, or activation of stress conditions such as
environmental or oxidative can also initiate autophagy (Ravindran et al. 2014). It
is basically a conserved cellular process that prolongs cell survival during stress
conditions by initiating the degradation of unnecessary cellular components like
nonfunctional macromolecules or damaged organelles into their basic units and
redirecting these molecules to be used by the cell. Apart from being a strategy of
replenishing essential components to the cell, autophagy has also been established as
an innate defense mechanism against various intracellular pathogens (like MTB),
viruses, fungi, bacteria, and protozoa (Deretic 2011; Nicola et al. 2012; Beale et al.
214 N. Khan et al.
Fig. 12.3 Mycobacterium tuberculosis infection evokes host stress response pathways
MTB infection induces ER stress and accumulation of unfolded proteins. ER stress sensors IRE1α,
PERK, and ATF6 get activated. Upon activation IRE1α dimerizes and splices XBP1 to a transcrip-
tion factor for molecular chaperones. It also activates NFκB through JNK pathway, which works as
a transcription factor for pro-inflammatory genes. Meanwhile, PERK upon activation dimerizes and
phosphorylates eIF2α and inhibits protein translation. Phosphorylated eIF2α induces apoptosis
signaling through CHOP. Apoptosis of MTB-infected macrophages releases ESAT6 that further
leads to apoptosis by elevating the levels of intracellular calcium in ER, which increases ROS and
ER stress
2014), by directing cytoplasmic components to lysosomal degradation (Yoshimori

and Noda 2008). Autophagy targets intracellular pathogens resulting in the engulf-
ment of cytoplasmic components by a double-membraned structure called the
autophagosomes, which could initiate from membrane sources including the endo-
some/Golgi system, ER-mitochondrial contact sites (Hailey et al. 2010; Hamasaki
et al. 2013), plasma membrane (Ravikumar et al. 2010), ER-Golgi intermediate
compartment (Ge et al. 2014), phospholipid precursors (Dupont et al. 2014), or
nuclear membrane in certain restricted conditions. It has also been implicated that
autophagy can modulate MHC class I-mediated antigen cross-presentation (Oliveira
and van Hall 2015). Different stress response pathways evoked during MTB infec-
tion are shown in Fig. 12.3.
Several regulators or inducers of autophagy have been reported, such as mamma-
lian target of rapamycin (mTOR), presence of microbes (Tattoli et al. 2012), the
TAB2/3-TAK1-IKK signaling axis (Criollo et al. 2011), or events downstream of
PRR and immune cytokine activation (Lee et al. 2007). MTB DNA also can act as a
signal to induce autophagy through the activation of TBK1 and stimulator of IFN
genes (STING)-dependent pathways which flag the pathogen with ubiquitin (Liang
et al. 2017). Induction of autophagy can be beneficial in the destruction of MTB as
the degradation of organelles releases antibacterial components that destroy the
pathogen (Alonso et al. 2007) and enhance peptide presentation (Jagannath et al.
2009). Gutierrez et al. first described in 2004 that a few of the bacilli are destroyed
through autophagy and this process is accelerated by factors such as IFN-γ by
facilitating phagosome-lysosome fusion and thus the maturation of
MTB-containing phagosomes to autophagolysosomes. Gutierrez et al. demonstrated
in a murine macrophage cell line stimulated with BCG that the LC3, an autophagy
marker, associated with MTB-containing phagosomes decreased the survival of
MTB in phagosomes (Gutierrez et al. 2004). Jagannath et al. examined the role of
autophagy on the processing of Ag85b, an immunodominant MTB antigen. The
authors found that rapamycin- and starvation-induced autophagy enhanced the
presentation of the antigen by APCs, macrophages, and DCs. The enhanced antigen
presentation through autophagy induction in MTB-infected macrophages and DCs
in turn deliberated in vivo protection in MTB-infected mice (Jagannath et al. 2009).
Therefore, autophagy can also be targeted to overcome the inhibition of
phagolysosome formation by MTB.
Autophagy is also been shown to be induced by anti-TB drugs that are being used
to treat active tuberculosis. Kim et al. showed that treatment of MTB-infected
macrophages with anti-mycobacterial antibiotics like isoniazid or pyrazinamide
robustly activated autophagy in the host cells and that this activation was necessary
for their effective drug action through the instigation of oxidative stress (Kim et al.
2012). These drugs subsidize to the formation of cellular and mitochondrial ROS
and thus facilitate the activation of autophagy through the recruitment of LC3 to
phagosomes (Huang et al. 2009). Treatment of MTB-infected bone marrow-derived
macrophages (BMDMs) with isoniazid increased the formation of LC3 punctate
which confirmed the autophagosome formation. The induction of autophagy further
orchestrated the production of inflammatory cytokines in the macrophages. Thus,
antibiotic-induced autophagy progressively targets the bacteria to more acidic
compartments and eventually for lysosomal degradation.
Albeit the detailed understanding about the host stress response pathways with
respect to MTB infection is at initial stages and requires more in-depth
investigations. Current studies highlight a critical role of host stress response
pathways in the regulation of innate as well as adaptive immune responses during
MTB infection.
12.5 Summary
TB is a prevalent bacterial disease that impacts majorly the developing countries

with its long duration and high cost of treatment. The host response to MTB is still
not completely understood, which hampers development of better vaccines and
therapeutics. Moreover, there are drawbacks in the dissemination of host response
toward MTB, as it is also dependent on genetic factors of the host as well as the
216 N. Khan et al.
pathogen. Even the reason behind latency and infection are not well understood.
Therefore, it needs a better understanding of the factors involved from both ends, the
host and the pathogen, to delineate the pathogenesis of MTB and to design better
vaccines and therapeutics. Consequently, new perceptions about ER stress, apopto-
sis, and autophagy in MTB infection from recent studies can be further explored as
targets for the development of a new and better TB vaccine.
Acknowledgments This work has been supported by the Department of Biotechnology, Govern-
ment of India [BT/PR8624/MED/29/798/2013], the Nano Mission Council, Department of Science
and Technology [DST No: SB/YS/LS-163/2013], and the University Grants Commission, Govern-
ment of India [MRP-MAJOR-BIOT-2013-40689].
Conflict of Interest The authors declare that the research was conducted in the absence of any
commercial or financial relationships that could be construed as a potential conflict of interest.
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Heat Shock Proteins in the Pathogenesis
of Mycobacterium tuberculosis 13
Prajna Tripathi and Janendra K. Batra
Abstract
Mycobacterium tuberculosis (M. tb), the causative agent of tuberculosis, is
responsible for immense global suffering taking nearly 1.5 million lives annually
(WHO 2016). About one-third of the world’s population is estimated to be
infected with this obligate pathogen and yet remains asymptomatic (Raviglione
and Sulis 2016). M. tb is highly adapted for survival in the extremely hostile
intracellular environment in host macrophages. The ability of M. tb to overcome
various host-induced proteotoxic stress conditions relies significantly on its
highly efficient chaperone network. Heat shock proteins (Hsps) form a special
class of the chaperone network and exhibit an orchestrated repertoire of stress-
sensing mechanisms. Hsps, found ubiquitously in most prokaryotes as well as
eukaryotes, are very well conserved across the species. In this chapter, we discuss
about the recent advances in understanding the myriad number of molecular
pathways that Hsps regulate, directly or otherwise in M. tb, which highlight the
association between Hsps and virulence determination in Mycobacterium.
Keywords
Host-pathogen interaction · Caseinolytic proteases · Virulence · Stress ·
Mycobacterium · Hsp
P. Tripathi
National Institute of Immunology, New Delhi, Delhi, India
J. K. Batra (*)
Department of Biochemistry, School of Chemical and Life Sciences, Jamia Hamdard, New Delhi,
Delhi, India
e-mail: jkbatra@jamiahamdard.ac.in

https://doi.org/10.1007/978-981-32-9413-4_13
222 P. Tripathi and J. K. Batra
Abbreviations
ATP Adenosine triphosphate

Clp Caseinolytic protease
IFNγ Interferon gamma
kDa Kilodalton
MHC Major histocompatibility complex
M. tb Mycobacterium tuberculosis
TB Tuberculosis
13.1 Introduction
Tuberculosis (TB), an infection caused by Mycobacterium tuberculosis (M. tb), is

responsible for immense global suffering taking nearly 1.5 million lives annually
(WHO 2016). About one third of the world’s population is estimated to be infected
with this obligate pathogen and yet remains asymptomatic (Raviglione and Sulis 2016).
M. tb is highly adapted for survival in the extremely hostile intracellular environment in
host macrophages. M. tb regulates its gene expression in order to endure a variety of
stresses produced by macrophages during infection. It can either persist in a quiescent
state or replicate actively within the host without being lethal to the infected host. The
outgrowth of bacilli is kept in check by the host defense mechanism which is well suited
to handle the invading pathogens. However, when the host defense system becomes
compromised, years after remaining latent, these bacilli may start resuscitating, enter
into an active phase, and may cause severe disease. A striking example of this is the case
of immune deficiencies taking place due to general health deterioration, undernourish-
ment, or HIV infection. Another reason for active tubercular infection is the emergence
of drug-resistant bacilli. The increase in occurrence of multidrug-resistant (MDR) and
extremely drug-resistant (XDR) strains of M. tb in the past decades has made the TB
treatment increasingly challenging (WHO 2016). Every year about 0.5 million cases of
MDR strains are reported (WHO 2016). The current treatment regimen for TB was
developed about 40 years ago with the introduction of four drugs, isoniazid, rifampicin,
pyrazinamide, and ethambutol, which formed the first line of defense. However, the
complexity of this regimen as well as the propensity of M. tb to develop drug resistance
has caused an urgent need for the development of novel therapeutics targeting other
pathways of the pathogen.
13.2 Mycobacterial Infection, Adaptation, and Survival Within

the Host
M. tb bacilli enter the host system primarily through respiratory route in the form of
aerosol, and once the bacilli reach alveoli, they are internalized via phagocytosis by
the alveolar macrophages. Previous studies have discovered that M. tb can also use a
13 Heat Shock Proteins in the Pathogenesis of Mycobacterium tuberculosis 223
number of receptors to access the macrophages (Ernst 1998; Aderem and Underhill
1999; Kang et al. 2005). The phagosomes, containing tubercle bacilli, make contact
with early endosomes of the host endocytic machinery but do not succeed in fusing
to lysosomes (Pieters and Gatfield 2002; Hestvik et al. 2005). The failure to fuse with
the lysosomes results in limited acidification of phagosomes, due to decreased
integration of the vacuolar proton ATPases (Sturgill-Koszycki et al. 1994). A
seminal study by Armstrong and Hart (1975) suggested that the ability of M. tb to
inhibit phagolysosomal fusion could be one of the many mechanisms that this
pathogen uses to sustain within the host. Some of the later studies reiterated that
M. tb infection leads to the arrest of phagosome maturation which is an important
strategy of these bacilli to survive within macrophages that are otherwise potent
microbicidal cells (Sturgill-Koszycki et al. 1994; MacMicking et al. 2003).
M. tb has been shown to survive as well as occasionally replicate within the
macrophages even after not being able to stall the phagosome-lysosome fusion
(Pethe et al. 2004; MacGurn and Cox 2007), and it can also endure highly acidified
environments (Gomes et al. 1999). Host macrophages attempt to contain these bacilli
within the phagolysosomes in the form of caseous granulomas. A granuloma can be
pathologically defined as an organized collection of differentiated macrophages with
a characteristic morphology. In case of mycobacterial infection, a granuloma is an
organized structure composed of a large number of multinucleated giant cells,
epithelioid cells with tight interdigitations linking adjacent cells, and foamy
macrophages surrounded on the outside by lymphocytes, neutrophils, fibroblasts,
dendritic cells (DCs), and other matrix components (Peters and Ernst 2003; Grosset
2003). A necrotic zone develops in the center of the granuloma due to the aggrega-
tion of acellular debris and is called “caseum” because of its milky appearance. The
rupture of this caseous center, a liquefied mass of bacilli, is crucial for the transmis-
sion of pulmonary tuberculosis (Flynn and Chan 2001). In some cases these
granulomas may become calcified and fibrotic, mostly containing dead bacilli
(Flynn and Chan 2001).
Though the formation of granuloma appears to be a host strategy to wall off or
eliminate mycobacterial infection, some bacilli still become successful in surviving
within the granuloma in a quiescent state. These lesions are widely assumed to be the
site of persistence of dormant or metabolically less active tubercle bacilli, where they
remain for years together. Somewhere in this deadlock between the host and the
pathogen, a shift may take place in favor of the pathogen, allowing its reactivation.
Intact M. tb bacilli have been recovered long after the infection from
phagolysosomes (Jordao et al. 2008) indicating that the bacteria must have adapted
to live in hostile environments. Persisting bacilli experience nutritionally deprived,
highly anoxic, and acidic environment in the macrophage. Infection with M. tb also
leads to stimulation of macrophages with IFNγ (Schaible et al. 1998; Via et al. 1998)
which in turn activates the production of reactive oxygen intermediates (ROI) and
reactive nitrogen intermediates (RNI).
Once the bacilli enter host macrophages, they encounter a plethora of stresses,
due to environmental fluctuation and host immune response, which include pH
stress, nutrient-depleted or carbon-starved environment, heat stress, and generation
of reactive oxygen species (ROS) by the phagosomal NADPH oxidases (Beste

2007; Axelrod 2008; Ehrt and Schnappinger 2009; Farhana et al. 2010; Butler
2010). Adaptation of M. tb to such extreme conditions is facilitated by a number of
mechanisms which include the manipulation of host cell response as well as
tolerance to the stress subjected by host defense system (Lowrie 1983; Chan
et al. 1992; Wayne and Sohaskey 2001; Firmani and Riley 2002; Farhana et al.
2010). To survive the lethal effects of the abovementioned antimicrobial molecules
released by activated macrophages, M. tb enters a stationary phase or a
non-replicating dormant phase (Wayne and Sohaskey 2001). The bacilli also
alter their gene expression pattern to cope with stressful conditions. In particular,
the induction of a set of evolutionarily highly conserved genes encoding heat shock
proteins (Hsps) plays a crucial role in conferring stress tolerance to microbial
pathogens (Lindquist and Craig 1988).
13.3 Heat Shock Proteins (Hsps): A Brief Introduction
Hsps, found ubiquitously in most prokaryotes as well as eukaryotes, are very well
conserved across the species. Hsps were first discovered as chromosomal puffs in
Drosophila melanogaster larvae when they were subjected to temperature stress
(Tissiéres et al. 1974). Hsps are significantly expressed in most organisms even
under normal conditions and take part in various fundamental cellular activities
along with facilitating protein quality control (Dobson and Karplus 1999). Classi-
cally, Hsps are defined as those proteins that bind to the exposed hydrophobic
patches on non-natively folded/aggregated proteins in a non-covalent manner
(Csermely et al. 1998). This underscores the role of many Hsps as molecular
chaperones, which are mostly ATP-dependent proteins that assist in quality control,
folding, and refolding of cellular proteins (Fig. 13.1). The conventional classification
of Hsps groups them into the following five different classes on the basis of their
molecular weights (Hartl and Hayer-Hartl 2002).
(i) Small heat shock proteins (sHSPs): They have low molecular weights but
form large oligomeric structures, usually composed of 24 subunits, and
function in an ATP-independent manner (Van Montfort et al. 2001;
Haslbeck et al. 2005). sHSPs are the least conserved class of Hsps and
generally contain an alpha-crystallin domain as their characteristic feature.
sHSPs are involved in protecting the partially unfolded or folded proteins
from aggregation (Haslbeck et al. 2005).
(ii) HSP60 family or GroE chaperone: The 60-kDa GroEL proteins are highly
conserved and form a barrel-shaped assembly composed of two
homoheptameric rings (Bukau and Horwich 1998). The double-ringed GroEL
chaperone interacts with its heptameric co-chaperone GroES, to constitute the
ATP-dependent GroEL-ES chaperone machinery (Hartl 1996; Sigler et al.
1998). This is the major chaperone machinery that assists protein folding in
bacterial cells (Houry et al. 1999).
Fig. 13.1 Functions of heat shock proteins. The schematic depicts various cellular activities
related to protein quality control where heat shock proteins play a key role
(iii) HSP70 family or DnaK chaperone: Members of the HSP70 family of

chaperones are conserved across all three surviving domains of life (Powers
and Balch 2013). DnaK plays a crucial role in the protein homeostasis network
by maintaining the native conformation of newly synthesized polypeptides as
well as existing cellular proteins, at the expense of ATP (Bukau and Horwich
1998). DnaK is known to associate with its co-chaperone Hsp40 (also known as
DnaJ in E. coli) and a nucleotide exchange factor GrpE, to form a complex
called KJE and perform chaperonic activity (Bukau and Horwich 1998;
Wickner et al. 1999).
(iv) HSP90 family: These are a conserved group of ATP-dependent cytosolic
chaperones. Hsp90 chaperone interacts with other chaperones and facilitates
the late-stage maturation and folding of proteins (Li and Buchner 2013).
(v) HSP100 family or caseinolytic proteases (Clps): The Clp proteins are an
integral part of the ATPases associated with diverse cellular activities (AAA
+) superfamily of proteins and, hence, contain the characteristic ATPase
domain(s) (Schirmer et al. 1996). The Clp proteins, ClpA, ClpX, and ClpC,
can act as stand-alone chaperones that prevent aggregation of substrates or
they can also function as the protein unfolding component of the chambered
Clp protease machinery by associating with the peptidase component ClpP,
which does not contain any ATPase domains. Clp proteins can prevent
protein aggregation and can also unfold the non-native proteins or
polypeptides for their subsequent degradation (Gottesman and Hendrickson

2000). At the structural level, Clp proteins form homohexameric ring-shaped
structure with a central cavity. They are broadly classified into two classes on
the basis of number of nucleotide-binding domains present in them. Each
monomer of the class I Clp proteins which includes ClpA, ClpB, and ClpC
possesses two nucleotide-binding domains (NBDs), whereas that of class II
Clp proteins that include ClpX and ClpY possesses only a single NBD
(Schirmer et al. 1996; Gottesman and Hendrickson 2000). ClpB is a unique
member of the Clp family as it does not take part in protein degradation;
instead it mediates the resolubilization of protein aggregates in association
with the KJE chaperone system (Weibezahn et al. 2003).
13.4 Hsps: A Suit of Armor
In order to survive within the host, a microbe must outcompete the environmental
fluctuations and perturbations, including the exposure to various cellular stresses. It
is well established that different environmental conditions can have profoundly
different impacts on the microbial pathogens, like Shigella, Yersinia,
Agrobacterium, fungi, viruses, etc., and could result in either elevated or repressed
growth and/or virulence of these organisms (O’Reilly and Zak 1992; Banta et al.
1998; Konkel and Tilly 2000; Klein and Tebbets 2007; Hatta et al. 2007). This
indicates that maintaining homeostatic balance is important for precise coordination
of microbial virulence and pathogenesis. It is therefore contingent upon the pathogen
integrating the environmental cues and initiating a fitting response to any stress-
related hostility. The prime adaptive response to elevation in stress levels includes
the induction of Hsps along with alternative sigma factors and other regulatory
proteins (Henderson et al. 2006).
The potential correlation between Hsps and bacterial pathogenesis or virulence
has been shown by many studies. In many bacteria, Hsps have been found to be
directly involved in pathogenesis. A 66-kDa Hsp has been shown to be involved in
the binding of Salmonella typhimurium cells to mucosal cells (Ensgraber and Loos
1992). One of the most striking examples of the involvement of Hsps in bacterial
pathogenesis is the infection of macrophages with Listeria monocytogenes. Ideally,
in an infected macrophage, the fusion of phagosome with an endosomal compart-
ment would lead to the digestion of the phagocytosed Listeria species. However,
Listeria averts this phenomenon by inducing a member of the Hsp100 family called
ClpP which mediates its release into the cytosol and hence multiplication (Gaillot
et al. 2000). The deletion of ClpP from the Listeria genome shows strong reduction
in bacterial virulence in mouse models of infection. The stress-induced expression of
ClpP has been linked to the rise in production of listeriolysin, a toxin responsible for
the phagosomal escape of Listeria (Gaillot et al. 2000). Additionally, another
member of the Hsp100 family, ClpC, is also shown to facilitate early phagosomal
escape of the bacteria (Nair et al. 2000) as well as its intracellular survival and
virulence in mice (Rouquette et al. 1996, 1998). ClpC in L. monocytogenes also
modulates various virulence factors, like InlA, InlB, and ActA, which are required
for adhesion and invasion into the host cells, while loss of ClpE leads to reduced
virulence (Nair et al. 1999). In Leishmania donovani, ClpB is required for full
amastigote development and also during the initial stages of mammalian infection
(Krobitsch and Clos 1999). Deletion of ClpB in Leptospira interrogans hampers the
general stress response and reduces virulence of the bacteria (Lourdault et al. 2011).
Porphyromonas gingivalis, the causative agent of periodontal disease, fails to
exhibit any phenotype with respect to bacterial adherence or invasion of cultured
human epithelial cells upon deletion of hsp90 from its genome (Sweier et al. 2003).
Similarly, the deletion of Hsp70 or DnaK from many bacterial pathogens, such as
Brucella suis, Campylobacter jejuni, and Salmonella enterica serovar typhimurium,
results in adverse effects on the intracellular growth and colonization of these
bacteria in mouse models of infection. Hsp70 has also been shown to be crucial
for the survival and virulence of malarial parasite, Plasmodium falciparum (Külzer
et al. 2012). The ability to evade internalization by phagocytes forms an important
aspect of pathogenesis of the human pathogen Staphylococcus. This phenomenon of
immune evasion has been reported to be regulated by the heat shock cognate protein,
Hsc70 (Hirschhausen et al. 2010). Cpn60, a protein of the HSP60 family, has been
reported to be crucial for epithelial cell invasion by Neisseria gonorrhoeae
(Tauschek et al. 1997; Du et al. 2005).
13.5 Contribution of Hsps Toward Pathogenesis and Virulence

of Mycobacterium
The ability of M. tb to overcome various host-induced proteotoxic stress conditions

relies significantly on its highly efficient chaperone network. Hsps form a special
class of the chaperone network and exhibit an orchestrated repertoire of stress
sensing mechanisms. Recent advances in understanding the myriad number of
molecular pathways that Hsps regulate, directly or otherwise in M. tb, highlight
the association between Hsps and virulence determination in Mycobacterium. Some
specific examples of the same will be discussed in this section.
The genome of M. tb encodes several heat shock proteins which mainly assist in
protein folding, refolding, degradation, and stress regulation. Some of the major
chaperone systems in Mycobacteria are discussed below.
(i) KJE complex which includes the Hsp70 homolog called DnaK, Hsp40
homologs called DnaJ1 and DnaJ2, and a nucleotide exchange factor called
GrpE. DnaK is reported to perform chaperonic activity as well as reactivation of
aggregated proteins in conjunction with ClpB (Lupoli et al. 2016). While both
the paralogs of M. tb DnaJ contain properties characteristic of a J-domain
protein and assist in chaperoning (Stewart et al. 2004; Lupoli et al. 2016),
only one of these is essential at a time for proper functioning of the KJE
machinery and survival of bacilli (Lupoli et al. 2016).
(ii) GroEL-GroES complex which includes the Hsp65 homolog called Cpn60.2 or
GroEL2 and its co-chaperone Cpn10 or GroES. Another paralog of GroEL2,
named GroEL1 or Cpn60.1, is known to exist in M. tb and is reported to be a
homolog of Hsp60. Both the GroEL proteins have ATPase activity. However,
unlike GroEL2, GroEL1 is nonessential for cell survival (Hu et al. 2008).
Though several studies speculate its function as a chaperonin (Sielaff et al.
2011) or in stress adaptation (Sharma et al. 2016; Bhat et al. 2017), the precise
biological role of GroEL1 still remains elusive.
(iii) HSP100 or the caseinolytic protease (Clp) machinery includes ClpX, ClpB, and
the two paralogs of ClpP, ClpP1, and P2 and of ClpC, ClpC1, and C2. All these
Clp proteins, except for ClpB, collaborate to form the Clp proteolytic machin-
ery (Raju et al. 2012; Schmitz and Sauer 2014), wherein homohexamers of
ClpC1 or ClpX act as the unfoldase subunit and the hetero-hexameric ClpP1P2
complex acts as the peptidase unit (Akopian et al. 2012). The Clp proteolytic
machinery is responsible for the turnover of proteins whose accumulation is
deleterious for the viability of mycobacterial cells. ClpC1 and ClpX are also
known to perform chaperonic activity on their own (Kar et al. 2008). ClpB acts
as a disaggregase and assists in bringing aggregated proteins back to their
native folded state. ClpB can perform stand-alone chaperonic activity as well
as it can associate with the KJE complex to enhance its activity (Lupoli et al.
2016).
(iv) Small heat shock proteins (sHSPs), which are ATP-independent chaperones,
form large oligomers and resolve partially folded proteins (Haslbeck et al.
2005). The most common member of this family is α-crystallin or Acr protein
which, due to its enhanced expression, is also considered as one of the markers
of non-replicating persistent phase of tubercle bacilli (Sherman et al. 2001;
Voskuil et al. 2003). M. tuberculosis genome contains two paralogs of the acr
gene, acr1 (previously referred to as acr or hspX) and an acr2 gene, whereas
M. leprae genome has a single homolog called acr3, and M. avium genome has
one acr2 and three functional acr3 genes.
Studies on the general stress response in M. tuberculosis and M. leprae indicate a

global upregulation of the operons which encode the Hsp70 chaperone protein,
DnaK; its co-chaperone Hsp40 protein, DnaJ1; and Hsp65 proteins, GroEL2 and
GroES (Stewart et al. 2001; Raman et al. 2001; Williams et al. 2007). The expression
of acr gene encoding Hsp20 protein has been found to increase in the TB bacilli by
as much as ~18–55 folds within 24 h in the mouse models of infection (Wilkinson
et al. 2005). In addition, Acr is reported to be important for the pathogenesis of M. tb
(Stewart et al. 2005). GroEL2, one of the major chaperones responsible for folding
linear amino acid chains into three-dimensional structures in M. tb, also acts as a
major adhesin for the binding of M. tb to monocytes (Hickey et al. 2009). Initial
investigations on the functionality of M. tb GroEL2 protein led to the discovery of its
pivotal role in controlling the pathogenesis of adjuvant arthritis – a rat model of
rheumatoid arthritis (van Eden et al. 1988). Since this seminal study, there have been
an enormous number of studies reiterating the potent immunomodulatory actions of
M. tb GroEL2. The host immune subversion phenomenon in M. tb is a major
contributor to its success as a human pathogen. In addition to its regular niche in
macrophages, these bacilli have also been known to interfere with the response of
DCs, which act as primary antigen-presenting cells in host. The cleaved form of
GroEL2, which predominates in mycobacterium, was recently shown to associate
with this phenomenon. In contrast to full-length GroEL2, the cleaved form of
GroEL2 was found to be poorly immunostimulatory and it dampened the process
of DC maturation (Georgieva et al. 2018). Additionally, it also contributes in
blocking the mitochondria-dependent apoptosis of infected macrophages which is
eventually implicated in the M. tb evasion of host innate immune response (Joseph
et al. 2017). The other paralog, GroEL1 or Cpn60.1, is implicated in the formation of
biofilms by mycobacterial bacilli and in caseous necrosis (Ojha et al. 2005). Biofilms
have long been associated with the robustness of M. tb and its pathogenesis. The
unique characteristics of mycobacterial biofilms impart resistance from environmen-
tal aggressions as well as antibiotics (Ojha et al. 2008; Esteban et al. 2008). Cpn10 or
GroES, the co-chaperone of GroELs, is shown to be responsible for the bone
resorption activity of M. tb (Meghji et al. 1997).
Hsp70 or DnaK is another major chaperone protein which assists in protein
quality control, and transposon mutagenesis studies have predicted Hsp70 to be
essential for cell viability and native protein folding in mycobacterial species (Griffin
et al. 2011; Fay and Glickman 2014). Interestingly, apart from its normal intracellu-
lar localization, DnaK has also been found to localize on the cell surface of virulent
M. tb bacilli which is indicative of its potential role in virulence (Hickey et al. 2009).
The chaperonic functions of DnaK are facilitated by either of the two homologs of
Hsp40 found in M. tb called DnaJ1 and DnaJ2 (Lupoli et al. 2016). The Hsp40
homologs have also been shown to be interacting partners of MBP64 protein (Chen
et al. 2011) which has long been implicated in virulence and pathogenesis of M. tb
(Harboe et al. 1996; Pym et al. 2002; Majlessi et al. 2005). Like GroEL2, Hsp40 also
exhibits immunomodulatory effects on rheumatoid arthritis patients (Tukaj et al.
2010), again indicating its potential in signaling within the host cells. Some of the
abovementioned Hsps are also immunodominant antigens found in the virulent
tubercle bacilli. Newer developments in the field of tuberculosis show that the
production of CC and CXC chemokines is responsible for the formation of granu-
loma (Méndez-Samperio et al. 2008). Hsp70 protein is reported to act as a major
inducer of these chemokines and hence aid driving the granuloma formation (Wang
et al. 2001).
Hsp22.5, another heat shock protein that gets activated under stress, has been
shown to be important for the survival of TB bacilli in murine models in the later
stages of chronic tuberculosis. Deletion of Hsp22.5 from the M. tb genome resulted
in altered transcription of some genes vital for ATP synthesis, protein synthesis,
dormancy, and lipid metabolism (Abomoelak et al. 2010).
ClpB was recently shown to function as an active disaggregase that resolves
protein aggregates in M. tb (Lupoli et al. 2016). Further, ClpB was shown to be
involved in sequestering and dividing the irreversibly oxidized proteins of stressed

mycobacteria within and between the cells (Vaubourgeix et al. 2015). Moreover, the
deletion of ClpB coding gene led to defects in growth and persistence of M. tb in
mice (Vaubourgeix et al. 2015).
A functional Clp protease has been shown to be essential for survival of
mycobacteria in vitro as well as during infection in mice (Raju et al. 2012).
Interestingly, ClpX, which is the substrate recognition subunit of Clp protease
assembly, has also been shown to interact with FtsZ and interfere with FtsZ
assembly (Dziedzic et al. 2010). FtsZ is a highly conserved homolog of the eukary-
otic protein tubulin and plays essential role in mycobacterial cell division (Romberg
and Levin 2003; Dziadek et al. 2003). FtsZ is known to localize at the mid-cell
division site in the form a Z-like ring which leads to initiation of the division process.
The Z-ring assembly is modulated in vivo and under antibiotic stress as a conse-
quence of interaction of ClpX with FtsZ (Chauhan et al. 2006; Dziedzic et al. 2010).
In addition, the GTP-dependent polymerization activity of FtsZ is also hindered
(Dziedzic et al. 2010). Thus, it becomes evident that ClpX also acts as an important
regulator of cell division and helps the tubercle bacilli cope with intracellular stress.
The Clp protease gene regulator, clgR, is reported to be strongly induced under
multiple stress conditions, such as hypoxia, redox stress, heat shock, SDS stress, etc.
(Stewart et al. 2001; Sherrid et al. 2010; McGillivray et al. 2014, 2015), which in
turn leads to a condition-dependent differential activation of downstream clp genes
that encode ClpB, ClpP1, P2, and ClpC1 (Mehra and Kaushal 2009; Barik et al.
2010; Personne et al. 2013). These reports further demonstrate that ClgR-regulated
transcriptional activation of clp genes is essential for M. tb to replicate within
macrophages and has important implications on the pathogenesis of mycobacteria.
As much as the heat shock proteins are shown to be critical for the viability and
virulence of M. tb, elevated expression of these proteins proves to be deleterious for
the organism in chronic phase of infection. Stewart et al. (2001) demonstrated that a
strain of M. tb which constitutively overexpressed Hsp70 because of deletion of the
transcriptional repressor HspR, though fully virulent in initial phase of infection,
showed significantly compromised persistence in subsequent stages of infection. In
the same study, it was reported that infection of mice with ΔhspR BCG strain led to
an enhanced induction of CD8+ IFN-γ-secreting T cells in the spleen indicating the
immunogenic potential of Hsps.
13.6 Therapeutic Potential of Hsps for TB Control
In order to thwart tuberculous infection timely, an effective diagnosis of the same is

of vital importance. At present, detection of tubercle bacilli in the body is based
mainly on two types of tests: Mantoux tuberculin skin test (TST) and interferon
gamma release assays (IGRAs) or the TB blood test. A positive patient is further
confirmed by sputum smear microscopy, which is usually followed by culturing the
bacteria. However, neither of these tests is able to discriminate between an active TB
disease and a latent TB infection (LTBI). The entire process of TB diagnosis is quite
consuming in terms of time and resources, and hence there is a need to develop
newer and faster diagnostic approaches and also to identify efficacious biomarkers of
TB. Since Hsps have already been elucidated to be involved in the progression of TB
infection, it is imperative to discuss their potential as biomarkers of the disease.
Hsps have recently started gaining attention as putative biomarkers of tuberculous
infections. Profiling of the cerebrospinal fluid of tuberculous meningitis (TBM)
patients has been carried out in various studies, where M. tb Hsp65, Hsp16, and
Hsp71 stand out as promising biomarkers (Kashyap et al. 2005; Mudaliar et al. 2006;
Tang et al. 2008). A similar profiling was done with the serum and sputum samples
of TB-positive patients, and it was found that fluctuations in the immune response of
host led to alterations in the profile of M. tb Hsps (Shekhawat et al. 2014). A
dramatic increase in the levels of Hsp16, Hsp65, and Hs71 was seen in patient
samples. A follow-up study, where monocytes were infected with the same human
clinical samples, was also found to corroborate the abovementioned findings. The
expression of Hsp16, Hsp65, and Hs71 was recorded to be as high as threefold in
only 48 h post infection (Shekhawat et al. 2014). These observations are also
indicative of the probable role of Hsps in host-pathogen interaction and ultimately
in altering the host immune response. Further, Hsp16 is reported to be highly specific
to latency-like conditions (Rajpal et al. 2011). Households with high risk of exposure
to TB also form an important part of the target group for interventions. Usually
household exposure to confirmed TB patients results in latent TB infection (LTBI)
which eludes normal diagnostic approach (Lienhardt et al. 2010). The levels of
Hsp16 have been shown to differentially increase in people with high risk of TB
exposure versus the low-risk exposure group or in the low-risk exposure group
versus the noninfected, healthy group of people (Shekhawat et al. 2016). The Hsp
profile in general was also found to be differential among the high-risk, low-risk, and
healthy subjects. Another study has shown sHsp18 as a major immunodominant
antigen resulting from M. leprae infection (Lini et al. 2008). Thus, the potential of
Hsps as biomarkers of the disease has been highlighted by both bedside and bench-
side investigations. They may also prove to be helpful in designing better diagnostics
for LTBI.
Apart from sharing the previously described diverse cellular functions like
protein turnover, stress tolerance, regulation of survival, etc., Hsps are also known
to perform a number of moonlighting functions owing to their secretion in the
extracellular milieu (Henderson et al. 2010). The extracellular secretion of Hsps is
a favorable feature as it enables the bacilli to influence the host immune response.
However, the exact mechanism of secretion is not yet clear. Released mycobacterial
Hsps bind to host cellular receptors and initiate an innate immune response, through
Toll-like receptors mostly, and result in secretion of pro-inflammatory molecules
(Lehner et al. 2004); hence they are also termed as “chaperokines” due to their dual
activity as chaperones as well as cytokines (Asea et al. 2000). Hsps, along with the
cargo of proteins that they carry, get processed and presented on host MHCs. Hsps
apparently act as a link between the innate and acquired immune responses and thus
have the ability to function as vaccine adjuvants as well (Segal et al. 2006).
Recombinant M. tb Hsp70, for example, shows significant adjuvant effect in
combination with recombinant outer membrane protein 31 (Dhakal et al. 2013).

Further, even significantly small quantities of Hsp70 bound peptides, approximately
120 pM, is sufficient to generate a cytotoxic T-cell response in vivo, while as high as
2000-folds of free peptide was unable to produce similar effect (Minton 2004; Javid
et al. 2004). Mycobacterial Hsp60 and Hsp70 have been used as effective carrier
molecules for conjugated vaccines. Cross-linking mycobacterial Hsp60 or Hsp70
with a synthetic peptide (NANP)40, an epitope from a major surface protein of
P. falciparum, and administering them without adjuvant to different strains of
mice as well as squirrel monkeys resulted in heightened production of anti-
(NANP)40 antibody (Lussow et al. 1991; Barrios et al. 1992; Perraut et al. 1993).
It has also been shown that mycobacterial Hsp70 dramatically enhances the immu-
nogenicity of HIV-1 gag p24 antigen (Suzue and Young 1996). Another promising
approach for control of TB is the use of DNA vaccines. Such a plasmid DNA
vaccine against TB was constructed by fusing mycobacterial Hsp65 and human IL-2
genes (Okada et al. 2007; Changhong et al. 2009). Administration of the Hsp-based
DNA vaccine to mice resulted in increased Th-1-type cellular response, and inter-
feron-γ and IL-2 were produced in greater amounts with a higher titer value of
antigen-specific anti-Hsp65 IgG2a. It is already established that the Th-1-type
response plays crucial role in controlling TB (Kim et al. 2000). The vaccine was
also able to provide significant protection against TB infection in challenge
experiments in mice. Another study also reports that M. tb Hsp65 DNA vaccine,
designed initially to control infection, can also have pronounced therapeutic effects
(Lowrie et al. 1999). Hsp16.3, a protein well documented to play key role in TB
persistence, has also been reported to have potential to be developed as new TB
vaccine component (Shi et al. 2009). Immunization of mice with Hsp16.3 or its
T-cell epitope was shown to produce antibodies that were significantly stronger and
much more specific as compared to the classical TB vaccine, BCG (Shi et al. 2009).
Colonization in the lungs and spleen of immunized mice was reported to have
dramatically reduced indicating the potential of Hsp16.3 as a prophylactic vaccine
candidate (Shi et al. 2009).
Taking into account the regulatory role of Hsps in physiological as well as
pathological conditions, they are beginning to emerge as potential candidates for
drug targeting and could be a breakthrough in the future. Many eukaryotic heat
shock proteins have already been targeted to treat cancer and numerous inhibitory
molecules of the same are under clinical trials (Taldone et al. 2009, 2014a, b).
Targeting Hsps is an active area of research currently in bacterial drug discovery
as well; however, targeting them against TB is still in its initial stages. Nevertheless,
development of inhibitors of GroEL/ES, Hsp90, GrpE, and DnaK in other bacteria
(Cellitti et al. 2009; Chapman et al. 2009; Minagawa et al. 2011; Abdeen et al. 2016)
gives us an indication that Hsps could also be potentially targeted in mycobacteria.
Mycobacterial Hsp70 or DnaK has been targeted with two of the terphenyls isolated
from the fungus Hypoxylon rickii, Rickenyl A and D, and they were reported to
function as inhibitors of Hsp70 (Mohammadi-Ostad-Kalayeh et al. 2017). They
compete with ATP for binding to DnaK in the range of 29 μM (Rickenyl D) and
49 μM (Rickenyl A). Structural differences in the bacterial and human Hsp70 protein
were shown to evidently affect the affinity of inhibitor molecules making them more
specific and selective for bacterial Hsp.
Clp proteins which belong to HSP100 family have been a major focus among the
Hsps for TB drug discovery efforts in the past decade. ClpP has been demonstrated
to be an unusual drug target, as both inhibition and hyperactivation of this gene can
lead to cell death. In Caulobacter crescentus, cyclic peptides have been found to be
able to bind to ClpXP-substrate complex, thereby preventing degradation of the
substrate and acting as bactericidal agents (Cheng et al. 2007). Compounds of
β-lactone family have also been reported to inhibit ClpP activity and attenuate
production of other virulence factors in S. aureus and L. monocytogenes (Gaillot
et al. 2000; Zeiler et al. 2011). ADEPs (acyldepsipeptides), on the other hand, are
known to activate the protease function of ClpP in M. tuberculosis (Brotz-Oesterhelt
et al. 2005). ADEPs also act against various other Gram-positive bacteria and their
multidrug-resistant isolates (Socha et al. 2010). ADEPs bind to ClpP on the same site
as the ATPase partner and lead to conformational changes in the oligomer which
eventually results in unregulated proteolysis and cell death (Lee et al. 2010; Li et al.
2010). Bortezomib, a peptidyl boronate antineoplastic drug, has been shown to
target ClpP1P2 of M. tb (Moreira et al. 2015). It also inhibits the M. tb 20S
proteasome system (Lin et al. 2008). Clp ATPase ClpC1 has been reported to be a
target for two natural inhibitor molecules, cyclomarin A (Schmitt et al. 2011) and
rufomycin (Gao et al. 2015), which act on the ClpC1-mediated ATP-dependent
proteolysis pathway. Cyclomarin A is a natural product that kills both replicating as
well as non-replicating mycobacteria. Another naturally occurring cyclic peptide
known as lassomycin has been demonstrated to have a similar spectrum of activity
against ClpC of M. tb resulting in stimulation of its activity without having any effect
on the proteolytic activity of ClpP. The end result of such activation is accumulation
of toxic proteins leading to death of the pathogen (Gavrish et al. 2014). Ecumicin,
another anti-mycobacterial natural peptide, also targets ClpC1 in a similar manner as
lassomycin (Choules et al., 2015). The peptides targeting ClpC1 display nanomolar
to micromolar potency against M. tb, with limited cytotoxicity to host cells. The
activities of the abovementioned molecules have been shown to be highly specific to
M. tb as they were found to be inactive against other Gram-positive bacteria (Gavrish
et al. 2014; Gao et al. 2015; Moreira et al. 2017). Moreover, several small organic
molecules displaying specific antimicrobial activity have also been identified against
E. coli ClpB (Martin et al. 2013).
13.7 Conclusion
Continuous surge in the drug resistance phenomenon in M. tb and problems due to

the current lengthy and complex treatment regimens have led to an urgent need to
develop new anti-tubercular agents. The crucial role of mycobacterial heat shock
proteins in the survival and maintenance of virulence of this pathogen makes them
attractive potential targets for newer therapeutic interventions. A great deal of work
has been done in the structure function analysis of heat shock proteins. Studies have
also implicated them in the pathogenesis of tuberculosis. Small organic molecules

inhibiting the in vitro activity of some of the mycobacterial Hsps have been designed
and characterized. Future in vivo studies will determine their real potential as
therapeutics against the TB bacterium. Inhibition of Hsps as a combination drug
therapy also offers a promising prospect. Further evaluation and understanding of
inhibitory molecules as well as their interaction with Hsps will hold immense
potential in designing novel and improved anti-tubercular drugs and vaccines.
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50:11001–11004
Endoplasmic Reticulum Stress: Importance
in Pathogenesis of Mycobacterium 14
tuberculosis
Tarina Sharma, Sonam Grover, Nasreen Z. Ehtesham,

and Seyed E. Hasnain
Abstract
Mycobacterium tuberculosis (M.tb), the cause of deadly disease tuberculosis, is
an opportunistic pathogen that primarily infects host alveolar macrophages. M.tb
has developed several mechanisms to persist in infected host cells and to dissem-
inate the disease. Its pathogenesis mainly depends on its competence to modulate
the host machinery for its own benefit. One of the cellular responses triggered by
M.tb is endoplasmic reticulum stress in infected macrophages, which eventually
disturbs the physiological functioning of the ER. Uncontrolled ER stress activates
IRE1, PERK, and ATF6 pathway to induce apoptosis of infected cells. Although
apoptosis is known to control and clear the infection in primary stages of
infection, in case of M.tb-infected ER stressed macrophages, apoptosis is able
to disseminate the pathogen during its advanced stages. M.tb-infected lung
granulomas are the preferential site of accumulation of apoptotic macrophages,
Tarina Sharma and Sonam Grover have equally contributed to this chapter.
T. Sharma
Molecular Infection and Functional Biology Lab, Kusuma School of Biological Sciences, Indian
Institute of Technology-Delhi, New Delhi, Delhi, India
S. Grover
N. Z. Ehtesham
S. E. Hasnain (*)

https://doi.org/10.1007/978-981-32-9413-4_14
242 T. Sharma et al.
thereby increasing the risk of disease dissemination. The present chapter will
describe the mechanism for ER stress response generated by known M.tb viru-
lence factors such as ESAT-6, HBHA, 38-kDa antigen, and PE_PGRS5. Future
insights to describe M.tb infection in respect of eliciting ER stress response-
mediated apoptosis and host interacting partners has the potential in identifying
novel targets for vaccination and drugs to combat the disease.
Keywords
Mycobacterium tuberculosis · Endoplasmic reticulum stress · Unfolded protein
response · Apoptosis · Oxidative stress · Calcium homeostasis
Abbreviations
ATF4 Activating transcription factor 4

ATF6 Activating transcription factor 6
bZiP Basic leucine zipper
CHOP C/EBP homologous protein
CREB cAMP response element binding
ER Endoplasmic reticulum
ERAD ER-associated degradation
ERSE ERS response elements
GRP78 78-kDA glucose regulated protein
HBHA Heparin-binding hemagglutinin antigen
Hsp70 Heat shock protein family
IP3R Inositol 1,4,5-triphate receptor
IRE1 Inositol requiring enzyme 1a
JNK Jun-N-terminal kinase
MAPK Mitogen-activated protein kinases
MCPIP MCP-1- induced protein
MPTP Mitochondrial permeability transition pore
NO Nitric oxide
PERK Protein kinase-like ER kinase
ROS Reactive oxygen species
SERCA Sarco-endoplasmic reticulum calcium ATPase
TB Tuberculosis
TRAF2 TNF-receptor associated factor 2
UPR Unfolded protein response
UPRE UPR element
XBP1 X-box binding protein 1
14 Endoplasmic Reticulum Stress: Importance in Pathogenesis of Mycobacterium. . . 243
14.1 Introduction
Tuberculosis (TB), caused by the opportunistic pathogen Mycobacterium tubercu-

losis (M.tb), remains the most common deadly disease. According to the 2017 WHO
TB report, 6.3 million new cases were reported with 1.3 million deaths in
HIV-negative people with an additional 3,74,000 deaths among HIV-positive
cases worldwide. Occurrence of multi-drug-resistant and extensive drug-resistant
cases has further worsened the scenario, which has become further complicated due
to HIV co-infections (WHO 2017).
M.tb, a diplomatic pathogen, is easily adapted in human host and employs
multiple strategies to survive and multiply inside macrophages. The pathogenesis
of TB depends on the interplay between host defense mechanisms and survival
strategies employed by the bacteria. Host mechanisms employed to clear the infec-
tion includes phagolysosome formation, apoptosis of host cells, generation of
immune responses, endoplasmic reticulum (ER) stress, etc. On one hand, as the
immune system tries to detect and clear the infection, the bacterium on the other
hand resists the immune response through several mechanisms that are the primary
cause of death during persistent infections. Once infected, M.tb secretes several
proteins in the macrophages for its survival in highly acidic phagolysosomal
compartments (Brodin et al. 2010). M.tb secretes several proteins, which are largely
in disordered state and cause the host cells to evoke an unfolded protein response
(UPR) (Ahmad et al. 2018; Grover et al. 2018). ER stress is one such response
generated by host cells to cope up with unwanted proteins produced by the pathogen.
On the contrary, M.tb employs this mechanism for its own survival as well. Thus,
back and forth interplay between these mechanisms such as ER stress-dependent
apoptosis defines the outcome of disease.
Endoplasmic reticulum stress is usually a mechanism provoked in response to
unfolded or misfolded proteins in ER. During some infectious diseases, the ER stress
response is generated by the interaction of pathogen proteins with ER stress sensor
proteins. As a consequence of the ER stress response, UPR is initiated and favors the
host to overcome the deleterious effects of stress accumulation. ER stress-mediated
response is a general phenomenon occurring in host cells; however in some cases
pathogens may employ this as a strategy to initiate signaling cascades leading to
disease pathogenesis. During different stages of infection, the ER stress response
leads to different outcomes either as a pro-host or pro-pathogen.
M.tb also employs various strategies to combat host responses generated upon
infection. An important factor in M.tb pathogenesis is induction of ER stress by M.tb
virulence factors. Some of the key M.tb virulence factors involved in ER stress are
ESAT-6 (Rv3875) (Choi et al. 2010), 38-kDa antigen (Rv0934) (Lim et al. 2015),
heparin-binding hemagglutinin antigen (HBHA) (Rv0475) (Choi et al. 2013), and
PE_PGRS5 (Rv0297) (Grover et al. 2018). Prolonged or uncontrolled ER stress
leads to apoptosis of the cell and clearance of stress responses. Previous reports have
shown that necrosis may favor the spread of mycobacteria, while apoptosis
facilitates to control the infection. Contradictory to this, more recent reports have
suggested a role for apoptosis in mycobacterial pathogenesis (Srinivasan et al. 2014).
Apoptosis, thought to play a significant role in defense mechanism employed by

macrophages to clear the infection, can also be hired by bacteria for its own survival
and dissemination. A role of ER stress-mediated apoptosis by live M.tb H37Rv in
bacterium survival has been demonstrated, where infection of macrophages with M.
tb initiates the ER stress and subsequent apoptosis of infected cells to disseminate the
bacterium (Lim et al. 2011).
14.2 Endoplasmic Reticulum Stress-Mediated Pathways

in Mycobacterial-Infected Macrophages
ER, an organelle containing chaperones and enzymes, is the site for protein folding
and transportation of proteins. Properly folded proteins from ER get modified in the
Golgi apparatus and are subsequently targeted to their destined sites in the cell
(Smith et al. 2011). Immature protein aggregates and misfolded proteins in ER are
targeted to ER-associated degradation (ERAD) machinery for clearance or by
autophagic degradation (Araki and Nagata 2012). Protein aggregation is a phenom-
enon in which misfolded or unfolded proteins form clumps and aggregates due to
their exposed hydrophobic amino acids. Protein aggregation in the ER lumen creates
a state of ER stress in cells, which contributes to pathological conditions in several
diseases such as diabetes mellitus, Alzheimer’s disease, Parkinson’s disease, athero-
sclerosis, and a few infectious diseases (Bhandary et al. 2013).
Mycobacterial infection also triggers ER stress (Lim et al. 2011), which leads to
disturbance in protein folding in infected cells. It results in the activation of UPR to
maintain regular functioning of the cells. Apart from performing protein folding and
transportation after protein synthesis, ER also serves as major reservoir of intracel-
lular Ca2+ ions, cholesterol steroids, and lipid synthesis. Depletion in calcium levels
from ER due to mycobacterial infection leads to misfolded and unfolded protein
accumulation. This accumulation of unfolded proteins results in ER stress and
subsequent apoptosis of infected cells. Induction of apoptosis in ER stress-dependent
manner by mycobacterial infection may either help in clearing the infection in early
stages or may allow the bacterium to disseminate through apoptotic bodies during
later stages of infection. An enhanced level of rough ER and smooth ER in
macrophages was observed when infected with the virulent strain of M.tb H37Rv
and attenuated strain of M.tb H37Ra, respectively (Saquib et al. 2015).
Four underlying mechanisms by host cells in response to ER stress are
(a) inhibition of protein synthesis to reduce protein aggregation; (b) production of
ER chaperones to increase protein folding; (c) activation of ERAD genes to enhance
ERAD machinery functioning; and (d) apoptosis of ER stressed cells (Yoshida 2007).
A mycobacterial-infected cell under uncontrolled ER stress activates signaling
pathways that subsequently direct the cells to undergo apoptosis. Failure to perform
this, host cells continue to harbor mycobacteria, thus facilitating its survival. The
three ER stress sensor proteins localized in ER are (a) protein kinase-like ER kinase
(PERK), a double-stranded RNA-dependent protein kinase; (b) inositol-requiring
enzyme 1a (IRE1a); and (c) activating transcription factor 6 (ATF6). Another
important factor in ER stress response is regulation of Ca2+ homeostasis. Disruption

in calcium levels affect the protein folding in ER stressed cells and induce apoptosis
(Cui et al. 2016).
14.2.1 IRE1a-Initiated ER Stress Response
IRE1a is a type I transmembrane sensor involved in activation of UPR for

maintaining physiological conditioning of cells. Generally, mammalian IRE1a is
involved in survivability of cells, but it is also known to degrade anti-apoptotic
miRNAs to induce apoptosis (Sano and Reed 2013). Its luminal domain forms
homo-dimers upon sensing ER stress, and its cytosolic domain gets auto-
phosphorylated stimulating the kinase activity. Due to this kinase activity, the basic
leucine zipper (bZiP) containing transcription factor, cAMP response element bind-
ing (CREB)/activating transcription factor (ATF) gets activated (Hassler et al. 2012).
X-box binding protein 1 (XBP1), a transcriptional activator interacts with UPR
element UPRE and ERS response elements (ERSE-I and ERSE-II) of promoter of
target genes. It thus induces the expression of other related proteins to reduce ER
stress. During M.tb infection, induction of ER stress leads to overstimulation of Toll-
like receptors (TLRs) in macrophages in an XBP1-dependent manner thereby
controlling mycobacterial infection (Lim et al. 2015; Martinon et al. 2010). RNase
domain of activated IRE1a interacts with TNF-receptor-associated factor 2 (TRAF2)
and forms the IRE1-TRAF2-ASK1 complex, which further stimulates the mitogen-
activated protein kinases (p38 MAPK) and Jun-N-terminal kinase (JNK) (Ron and
Hubbard 2008). M. kansasii-infected macrophages have undergone apoptosis
through an ER stress-dependent IRE1a/ASK1/JNK cascade (Lim et al. 2015).
14.2.2 The PERK Pathway
PERK, a serine/threonine protein kinase, is a type I transmembrane protein. Its

destined function is to attenuate the protein synthesis machinery to reduce protein
aggregation in ER stressed cells. PERK/eIF2a/CHOP pathway is activated by
macrophages in response to M.tb infection and 38-kDa antigen (Lim et al. 2015).
Upon sensing ER stress, PERK releases BiP (chaperone molecule), which afterwards
gets oligomerized and auto-phosphorylated. PERK when activated phosphorylates
eukaryotic initiation factor (eIF2a) at Ser51 residue. This phosphorylation inhibits
the initiation complex of translational machinery thus haltering the new polypeptide
synthesis. It reduces the global protein accumulation and the burden on ERAD
process for protein folding thereby maintaining physiological conditioning of the
cell (Bertolotti et al. 2000).
Activating transcription factor 4 (ATF4) is a transcription factor that belongs to
the CREB protein family. Selective induction of ATF4 mRNA by eIF2a leads to its
overexpression, which in turn induces the transcription of UPR transcription factors
and ER stress response folding proteins. These factors then translocate to the nucleus
for initiating ER stress response. ATF4 also initiates the transfer of activating
transcription factor 6 (ATF6) from ER to Golgi apparatus.
C/EBP homologous protein (CHOP) is another component of ER stress-mediated
apoptosis. During M.tb infection, CHOP levels considerably increase in the granu-
lomatous regions (Seimon et al. 2010). Infection studies have shown that M.tb
exploits the reduced levels of CHOP due to a decreased eIF2a phosphorylation for
its own survival. It indicates that M.tb modulates host cell ER stress response for its
own survival (Lim et al. 2011). In TB granulomas, ER stress is evident in specific
areas of accumulated apoptotic macrophages (Seimon et al. 2010). Initially, overall
protein synthesis was halted by PERK using eIF2a to promote cell survival. Later,
due to an uncontrolled ER stress caused by M.tb infection, downstream CHOP is
activated by PERK to induce apoptosis.
14.2.3 ATF6 Induces ER Stress Response
ATF6, a 90 kDa ER transmembrane protein, is translocated from ER to Golgi

apparatus during conditions of ER stress in the cell. Its N-terminal region consists
of the CREB/bZip transcription factor. ATF6a and ATF6b are two subtypes of ATF6
in mammalian cells. It gets activated in response to ER stress and cleaved to a 50 kDa
protein fragment by proteolytic cleavage at its N-terminal site (Shen et al. 2002).
Cleaved fragments get translocated to the nucleus for promoting transcription of ER
stress response-related genes such as cAMP response element (CRE), ERSE-I,
ERSE- II, and UPR element (UPRE). Binding of ATF6 to XBP1 induces
UPR-related proteins expression. These proteins altogether relieve the stress from
cells by reducing misfolded protein accumulation (Wang et al. 2000).
14.2.4 Chaperones in ER Stress Response
Unfolded protein response is activated to protect cells against adverse effects of ER

stress. ER provides environment of proper protein folding by the presence of many
enzymes and chaperones. Appropriate protein folding is an important phenomenon
in maintaining physiological functioning of cells as only correctly folded proteins
can be modified and translocated to the destined location (Smith et al. 2011). ER
chaperones play a major role in protein synthesis regulation as well as in cell death
induction (Tabas and Ron 2011). One of the molecular chaperones from the heat
shock protein family (Hsp70) also known as GRP78/BiP is employed in rescuing
cells from stressed conditions. BiP functions by participating in folding and assem-
bly of proteins as well as targeting the misfolded/unfolded proteins towards degra-
dation by 26S ubiquitination pathway. Bip is also a resident of mitochondria and
nucleus apart from ER and upregulated in tumor cells (Ortiz and Cardemil 2001;
Zhang et al. 2010). Bip is found to be overexpressed under various stress conditions
such as depleted calcium/oxygen levels and glycopenia. Upregulated expression of

Bip during M.tb and other mycobacterial species has also been reported in several
studies (Choi et al. 2013; Lim et al. 2011).
Under resting conditions, inactive Bip is generally found to be non-covalently
associated with ER stress receptors such as IRE1, PERK, and ATF6. On sensing
stress conditions, Bip dissociates from the stress receptors and UPR is triggered.
Dissociation of Bip from IRE1, PERK, and ATF6 leads to signaling cascades
required for neutralizing the deleterious effects of ER stress and brings the ER
functions back to normal (Harding et al. 1999). Few of the mycobacterial proteins
such as ESAT-6, HBHA, and 38-kDa antigen are known to upregulate expression of
ER chaperones, thus inducing ER stress responses. Increased expression of the
pro-inflammatory cytokine, MCP-1-induced protein (MCPIP) in response to Mtb
38-kDa antigen, leads to ROS generation and subsequent misfolding of proteins.
These events trigger Bip to be released from ER stress sensors and initiate protein
folding. Other ER chaperones including CHOP and activated phosphorylated eIF2
has also been shown to be expressed by cells in response M.tb 38-kDa Ag (Lim et al.
2015).
14.2.5 Oxidative Stress in ER Stress
ER provides a suitable oxidative environment for the protein folding process. A

change in oxidative environment and intracellular calcium levels induce the produc-
tion of reactive oxygen species (ROS) in cells. ROS production in stressed cells is
linked to protein folding competence of ER. ROS and nitric oxide (NO) generation
are not only the consequences of ER stress but also stand as an integral process
during ER stress. ROS may be generated as a by-product in electron transfer
reactions or because of reduced glutathione (GSH) depletion during protein
misfolding (Santos et al. 2009).
Mitochondrial ROS generation also tunes the downstream effects during ER
stress. Mitochondrial ROS accumulation due to depleted GSH can enhance ER
stress and also leads to cell mortality during persistent ER stress (Yoon et al.
2011). The electron transport chain and mitochondrial phosphorylation plays an
important role in ROS generation. Mitochondrial respiration interference in cells
reduces ROS accumulation (Bravo et al. 2011). Cells devoid of cytochrome C gene
have mitigated response in context to UPR induction by hypoxia (Liu et al. 2008).
During ER stress induction, ER and mitochondria appear to function together to
cope up this situation. Firstly, ER and mitochondria lie in close vicinity to each other
that suggests a physical interaction between the two organelles (Raturi and Simmen
2013). Secondly, Ca2+ levels affect the mitochondrial membrane potential and ROS
production (Bravo et al. 2011). Thirdly, there is expression of Lon protease and NIX
(regulating mitochondrial membrane potential and ER Ca2+). In response to these
signaling molecules, the mitochondrial permeability transition pore (MPTP) gets
opened (Chen et al. 2010).
Enhanced oxidative stress in cells leads to an inflow of Ca2+ from the extracellular
environment and also an outflow from the ER. Ca2+ ions henceforth move to the
mitochondria and nuclei stimulating the mitochondrial process and further ROS
generation (Murphy 2009). An increase in ROS levels in the mitochondria in return
proliferates the cascade of Ca2+ release from the ER (Moserova and Kralova 2012)
and subsequent deterioration in ER functioning. Thus, the interplay between ER and
mitochondrial-dependent oxidative stress decides the overall fate of stressed cells.
M.tb live infection has been shown to induced ROS elevation during ER stress
response forcing the infected cells to undergo apoptosis. This ER stress-mediated
apoptosis has been linked with mycobacterial survival (Lim et al. 2011). ER stress-
mediated apoptosis of M. kansasii SM-1-infected cells (Lim et al. 2013) and M.tb
ESAT-6-stimulated cells (Choi et al. 2010) majorly depends on significant ROS
production as a consequence of ER stress.
14.2.6 Calcium Homeostasis in ER Stress
ER, a major reservoir of Ca2+ inside cells, maintains calcium homeostasis for proper
folding during stressed conditions. An alteration in their levels leads to apoptosis of
the stressed cells due to improper folding or uncontrolled stress. Release of Ca2+
from ER in response to M.tb infection has been shown to reduce the protein-folding
capacity of ER in stressed cells. This reduction in Ca2+ levels is linked to CHOP
activation by M.tb infection. Another consequence of Ca2+ release is its entry in
mitochondria and subsequent activation of oxidative stress in the form of ROS and
NO production (Sah et al. 1999). Altogether these effects allow the stressed cells to
undergo apoptosis (Chen et al. 2013; Lim et al. 2015). M.tb infection leads to an
increase in Ca2+ results in ROS-mediated apoptosis. Activation of CHOP by M.tb
infection initiates the disintegration of mitochondrial membrane leading to release
Ca2+. The release of cytochrome C from mitochondria then causes the apoptosis of
cells. Calcium-regulated proteins will also get affected by this increase in Ca2+
levels. Calpain is a proteinase, which gets activated by Ca2+, and induces apoptosis
by activating caspase12 expression. ESAT-6 of M.tb has been shown to induce
calpain activation, which subsequently leads to apoptosis in caspase12-dependent
manner (Choi et al. 2010). Calreticulin, a glycoprotein chaperone of ER, is Ca2+
binding protein that preforms folding of unfolded proteins. The overexpression of
calreticulin sensitizes the cells to undergo apoptosis (Ferri and Kroemer 2001).
14.3 Mycobacterial Proteins Involved in ER Stress
Mycobacterial pathogenesis is linked to ER stress in host cells (Cui et al. 2016).

Macrophages infected with live M.tb H37Rv have increased levels of rough ER
(Saquib et al. 2015). The ER stress-mediated pathway in M.tb-infected macrophages
has proved to be a survival advantage to the pathogen (Lim et al. 2011). Although
apoptosis has been thought to be a protective mechanism against the pathogen, it
may not prove beneficial in later stages of M.tb infection. At the sites of TB
granulomas, where an accumulation of apoptotic macrophages has been observed,
an increased level of ER stress is evident (Seimon et al. 2010). During M.tb
infection, this ER stress-mediated apoptosis may favor the mycobacteria to
disseminate.
Few of the mycobacterial proteins listed in Table 14.1 have been identified to be
involved in ER stress response and ER stress-mediated apoptosis (Fig. 14.1). The
mode of action for generating ER stress responses by M.tb proteins are illustrated in
Fig. 14.2.
Table 14.1 Mycobacterial proteins involved in ER stress-mediated cellular responses

Sl. Mycobacterial
No. protein ER stress response References
1 ESAT-6 eIF2a/ATF4/CHOP upregulation Choi et al. (2010)
Enhanced Ca2+ and ROS levels
Apoptosis
2 HBHA CHOP upregulation Choi et al. (2013)
Apoptosis
3 38-kDa antigen PERK/eIF2a/CHOP upregulation Lim et al. (2013)
Apoptosis
TLR-2/4 dependent
4 PE_PGRS5 GRP78/GRP94 and CHOP/ATF4 Grover et al.
upregulation (2018)
Enhanced Ca2+, ROS, and NO levels
Apoptosis
TLR-4 dependent
Fig. 14.1 Endoplasmic stress-mediated apoptosis by proteins of Mycobacterium tuberculosis

Fig. 14.2 Mycobacterium tuberculosis evokes endoplasmic reticulum stress-mediated response

and apoptosis by ESAT-6, HBHA antigen, 38-kDa antigen, and PE_PGRS5 proteins
14.3.1 ESAT-6
ESAT-6 is an important virulence factor of M.tb involved in the early phases of TB

pathogenesis. ESAT-6, an important candidate for vaccine development, is known to
induce Th-1 immune responses (Wang et al. 2009). M.tb secretes ESAT-6 into
culture filtrates during early growth.
Human macrophages and epithelial cells stimulated with ESAT-6 antigen have
been shown to induce ER stress response and apoptosis. CHOP and 78-kDA
glucose-regulated protein (GRP78) and CHOP are known to be associated with
ER stress, and their expression has been known to be increased after ESAT-6
stimulation. ESAT-6 stimulation also upregulates the expression of ATF4 and
eIF2a phosphorylation in macrophages. Thus, ESAT-6 mediates the ER stress
response by eIF2a/ATF4/CHOP pathway. Apart from this pathway, ESAT-6 has
also shown to increase intracellular Ca2+ levels and ROS production in
macrophages. ER stress has been known to induce caspase12 activation for
promoting apoptosis in mammalian cells. Macrophages undergo apoptosis in a

caspase12-, caspase4-, and caspase3-dependent manner by mycobacterial ESAT-6
antigen. Also, ASK1-JNK pathway is involved in the ER stress response leading to
apoptosis related to ER stress response. ESAT-6 antigen, secreted from M.tb,
induces ER stress-induced apoptosis through the CHOP/ASK1/JNK-mediated path-
way (Choi et al. 2010).
14.3.2 HBHA Antigen
HBHA antigen is one of virulence factor of M.tb involved in pathogenesis and

infectivity. It is a 28 kDa, methylated surface protein (Parra et al. 2004). ROS and
NO production by ER stressed cells are associated with apoptosis and other down-
stream stress responses (Gotoh and Mori 2006).
HBHA antigen has been reported to increase cytosolic Ca2+ levels with an
enhanced ROS levels and promote ER stress-mediated apoptosis by inducing
CHOP-dependent caspases (Sohn et al. 2011). HBHA also leads to CHOP-
dependent expression of IL-6 and MCP-1 via Nf-kB activation. All these have
shown the role of ER stress in HBHA antigen-mediated apoptosis (Choi et al. 2013).
14.3.3 38-kDa Antigen
One of the most important M.tb antigen is the 38-kDa phosphate transport protein
(PstS-1) antigen, secreted by M.tb cultures (Harboe and Wiker 1992). The 38-kDa
antigen is shown to elevate the expression of the pro-inflammatory cytokine MCP-1-
induced protein (MCPIP). MCPIP subsequently increases ROS generation enhanc-
ing unfolded protein accumulation. Bip is then released from IRE1, PERK, and
ATF6 and binds misfolded proteins to reduce ER stress. The 38-kDa antigen-
induced CHOP production is reduced in case of JNK pathway inhibition. The p38
MAPK inhibition leads to activation of JNK and enhanced expression of CHOP by
38-kDa antigen stimulation. Suggested evidence points towards the role of 38-kDa
antigen in PERK/eIF2a/CHOP pathway activation during ER stress response (Lim
et al. 2015). The 38-kDa antigen provokes the ROS production and ER stress
response by ERO1a, which normalizes Ca2+ levels by the Sarco-endoplasmic Retic-
ulum Calcium ATPase (SERCA) inositol 1,4,5-triphate receptor (IP3R) (Ferri and
Kroemer 2001).
14.3.4 PE_PGRS5
Ten percent M.tb genome codes for PE/PPE/PE_PGRS proteins exclusively present
in pathogenic strains of mycobacteria (Akhter et al. 2012). Dissimilarities in
PE/PPE/PE_PGRS proteins from virulent H37Rv and avirulent H37Ra strains of M.tb
are responsible for virulence competence of the strain (Kohli et al. 2012). One of the
proteins of this family is PE_PGRS5 encoded by the Rv0297 gene, which has been
shown by transcriptomic studies to be expressed in granulomas in later stages of
infection (Kruh et al. 2010). The PGRS domain is highly disordered and includes
putative ER retention signals, which assist in its localization to the
ER. Rv0297PGRS leads to the activation of UPR and ER stress markers GRP78/
GRP94 and CHOP/ATF4, and subsequent disruption in intracellular Ca2+ levels
(Grover et al. 2018). Alteration of calcium levels leads to the production of ROS
(Hasnain et al. 1999), and ROS generation is related to the UPR pathway. ROS
production and oxidative stress are important components of the ER stress response.
Rv0297PGRS has also been shown to increase ROS and NO levels in macrophages.
The final ER stress response induced by PE_PGRS5 forces the cells to undergo
apoptosis via caspase8 activation. The role of TLR-4 in ER stress-mediated apopto-
sis by Rv0297PGRS has been evident (Grover et al. 2018).
14.4 Conclusion
ER is a site for folding and transportation of synthesized proteins. It also serves as

major site for storage of intracellular Ca2+. Infectious diseases may induce ER stress
response and aid in apoptosis of cells to clear the infection. In case of mycobacterial
infections, ER stress response is mediated through several virulence factors of M.tb.
Several of the M.tb proteins after interacting with ER stress sensor proteins of host
cells initiate the cascade of events leading to the unfolded protein response. UPR
serves to control or reduce the ER stress-related damage to maintain physiological
functioning of host cells. Upon prolonged or uncontrolled ER stress, the response
generated transforms into apoptosis, which subsequently helps the host to clear the
infection. In early stages of mycobacterial infection, apoptosis may help in clearing
the infection. But in later stages of infection, ER stress-mediated apoptosis may
favor the Mycobacterium to disseminate the disease. In M.tb-infected fibrocaseous
lung granulomas, apoptotic macrophages accumulate at sites of ER stress. Apoptosis
in infected foamy macrophages leads to caseum accumulation and progression of
disease pathogenesis. M.tb, a smart pathogen, modulates the host responses for its
own benefit and employs the host machinery to transmit the disease. Thus, ER stress-
mediated apoptosis may not always prove beneficial to the host and may rather aid in
pathogen survival and dissemination of disease. There is a need to investigate M.tb
proteins involved in the molecular mechanisms employed for mediating ER stress
response or apoptosis to target the pathogen. The host partners involved in these
survival strategies would also need to be elucidated in depth to provide insights for
the development of host directed interventions against the disease.
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Toxin-Antitoxin (TA) Systems in Stress
Survival and Pathogenesis 15
Ashutosh Kumar, Anwar Alam, Pranami Bharadwaj,
Sharmistha Tapadar, Mamta Rani, and Seyed E. Hasnain
Abstract
Mycobacterium tuberculosis (M.tb), by virtue of its ability to evolve, has devel-
oped mechanisms that enable it to modulate its growth through regulation of
replication, transcription, translation, generation of heterogeneous population of
persister cells, etc. for survival in different stressful environment during its
infection cycle. Toxin-antitoxin (TA) systems are ubiquitous in prokaryotic
genomes that enable them to survive in various unfavourable conditions. A
toxin protein may inhibit the growth, whereas an antitoxin may neutralize the
effect of toxin in different ways. TA systems are involved in stress adaptation,
antimicrobial tolerance or resistance, modification in the physiological state of
organisms, biofilms formation, growth regulation for survival, plasmid
maintenance, anti-phage activities, virulence, and programmed cell death.
Ashutosh Kumar and Anwar Alam have equally contributed to this chapter.
A. Kumar · P. Bharadwaj · S. Tapadar

Department of Microbiology, Tripura University (A Central University), Agartala, Tripura, India
A. Alam
M. Rani
Plant Microbe Interactions Laboratory, National Institute of Plant Genome Research, New Delhi,
Delhi, India
S. E. Hasnain (*)

https://doi.org/10.1007/978-981-32-9413-4_15
258 A. Kumar et al.
Environmental microorganisms express a wider repertoire of TA systems as

compared to intracellular human pathogens due to a higher probability to encoun-
ter different environmental stresses within their ecosystem. However, the pres-
ence of high level of TA systems in M.tb is due to the fact that M.tb has to endure
several types of stresses including acidic, hypoxic, oxidative, and immune sur-
veillance within the host for its survival. TA systems are also present in patho-
genic bacteria infecting plants. Based on the mechanism of action, different types
of TA systems are classified within the microorganisms. Recently, genes related
to type II TA systems have been proposed to be useful in genotyping of tubercu-
losis caused by different strains of M.tb.
Keywords
Toxin-antitoxin systems · Mycobacterium tuberculosis · Stress survival · Growth
regulation · Drug tolerance
Abbreviations
ATP Adenosine triphosphate

DATIN Dormancy-associated translation inhibitor
DNA Deoxyribonucleic acid
DR Direct repeat
E. coli Escherichia coli
IS Insertion sequence
mRNA Messenger RNA
MTBC M.tb complex
PCD Programmed cell death
PSK Post-segregational killing
SNPs Single-nucleotide polymorphisms
sRNA Small regulatory RNAs
TA Toxin-antitoxin
TAC Toxin-antitoxin-chaperone
Vap Virulence-associated protein
VNTR Variable number tandem repeats
15.1 Introduction
Toxin-antitoxin (TA) systems in bacteria were first recognized as plasmid-borne loci

which help in plasmid maintenance through elimination of daughter cells lacking TA
encoding plasmid (Guglielmini and Melderen 2011). A set of linked genes, together
encoding a protein ‘poison’ and a corresponding ‘antidote’, forms the TA system
(Gerdes 2000). The TA systems present on plasmids make sure that only the
15 Toxin-Antitoxin (TA) Systems in Stress Survival and Pathogenesis 259
daughter cells inheriting the plasmid survive after cell division. In daughter cells
devoid of plasmid, unstable antitoxin is degraded while the stable toxic protein kills
the new cell, and this phenomenon is known as ‘post-segregational killing’ (PSK).
TA systems are present in multiple copies in prokaryotes (Yamaguchi et al. 2011).
Various microbial genome analyses have comprehensively highlighted the diversity
in the distribution of TA systems.
Previous studies have shown that the genomes of Nitrosomonas europaea,
Sinorhizobium meliloti, and Mycobacterium bovis contain more than 50 presumptive
TA systems, whereas Rickettsia prowazekii, Bacillus subtilis, Campylobacter jejuni,
etc. contain no or very few TA systems (Pandey and Gerdes 2005; Sevin and Barloy-
Hubler 2007). However, there is little consensus to prove correlation between the
number of TA systems and the growth rate of the members within a phylum.
Additionally, diversity in the distribution of TA systems among different isolates
of the same species is also observed.
Detailed study of phylogenetic patterns of TA loci in several prokaryotic
genomes suggests presence of multiple TA loci in free-living prokaryotes and few
or no TA loci in obligate intracellular prokaryotes (Pandey and Gerdes 2005). TA
loci are beneficial to organisms that confront stressful environment. TA systems,
also referred to as junk, are considered to be constituents of plasmids and have been
retained within the cells due to their addictive nature (Kroll et al. 2010). Some toxins
act as general repressors of gene expression, while others are more specific in
regulating gene expression (Engelberg-Kulka et al. 2006; Pimentel et al. 2005).
Some TA systems that act as bacteriostatic toxins play a key role in growth regula-
tion and may restrict growth rather than kill the host cell (Diago-Navarro et al. 2010).
‘Persisters’ are slow-growing population of cells, which can survive stress and later
grow into actively dividing cells when the environment is favourable (Kussell et al.
2005). It has been demonstrated that an imbalance between the level of toxin and its
antitoxin due to overexpression or mutations in either of them results in high
persistence (Fridman et al. 2014; Korch and Hill 2006). Interruption of transcription
and translation machinery of host cells due to bacteriophage may also trigger
activation of TA system that in turn limits phage replication, termed as antiphage
mechanism (Hazan and Engelberg-Kulka 2004).
M.tb faces different stresses in its pathogenesis and possesses several proteins such
as two component systems, sigma factors, TA systems, acid response, halophilic
proteins, etc. for its survival (Kumar et al. 2018). As compared to other mycobacteria,
M.tb shows presence of a significant number of TA systems in its genome, which
during the state of persistence are induced by active toxins, which may largely
contribute towards its pathogenesis (Ramage et al. 2009). Transcriptomic analyses
of antibiotic-induced M.tb persisters showed that about 10 TA systems were signifi-
cantly upregulated, pointing to the importance of TA system in M.tb persistence
(Keren et al. 2011). Interestingly, M.tb possesses abundant number of TA loci, while
M. leprae has none, possibly due to the fact that M. leprae has evolved from M.tb
through reductive evolution (Ramage et al. 2009; Cole et al. 2001). The presence of
TA system provides an evolutionary edge to M.tb in terms of aiding its survival in
both extra- and intracellular conditions as compared to M. leprae which can survive
260 A. Kumar et al.
only as obligate intracellular pathogen. Similarly, obligate intracellular

microorganisms such as Rickettsia and Buchnera spp. have either very few or no
TA loci, although several exceptions are also in existence (Pandey and Gerdes 2005;
Leplae et al. 2011). Analyses of bacterial gene sequences by Shao et al. (Shao et al.
2011) conclusively point to the presence of TA systems in symbiotic bacteria and
overrules previous studies by Pandey et al. that reported absence of TA systems in
symbiotic bacteria (Pandey and Gerdes 2005; Shao et al. 2011).
15.2 Classes of Toxin-Antitoxin Systems
The TA loci are classified into different groups such as vapBC, parDE, relBE, ccd,
phd/doc, mazEF, and higBA due of differences in mechanism of action (Gerdes et al.
2005). In M.tb, majority of TA systems belong to the class of VapBC (virulence-
associated protein) (Gerdes and Maisonneuve 2012). VapC induces dormancy by
suppressing translation and induction of vapB transcription which later leads to
revival of cells (Winther and Gerdes 2009). It has been shown recently by deletion
and overexpression studies that some members of the VapBC TA systems of M.tb
are involved in bacteriostasis, morphological changes, growth arrest, and mycobac-
terial pathogenesis (Agarwal et al. 2018). M.tb VapBC30 system has been shown to
be involved in growth regulation through ribonuclease activity (Deep et al. 2018). M.
tb toxin VapC30 inhibits the growth of Escherichia coli (E. coli) when expressed
without its cognate antitoxin VapB30. There is no effect of M.tb VapC30 on E. coli
when co-expressed with M.tb VapB30. M.tb VapC30 degrades RNA molecules that
are magnesium and manganese ion dependent (Lee et al. 2015).
M.tb MazF toxin members, when expressed in E. coli or Mycobacterium
smegmatis, affect their growth (Gupta 2009; Zhu et al. 2006). The overexpression
of MazF3, MazF6, and MazF9 of M.tb in Mycobacterium bovis BCG induces
bacteriostasis (Tiwari et al. 2015). M.tb MazF toxins are also involved in the drug
tolerance, virulence, and stress adaptation (Tiwari et al. 2015).
The RelBE system is among the most characterized TA systems that bind with the
A site of the ribosome and affect protein synthesis by cleaving the mRNAs prefera-
bly between the second and third nucleotides of the termination codon (Pedersen
2003). In contrast, RelE binds to initial coding region and cleaves the first 100 codons
of mRNA and inhibits growth (Hurley et al. 2011). Variety of stress conditions such
as nitrosative stress, oxidative stress, and antibiotic stress affect the transcript
profiles of RelE toxins of M.tb. The overexpression of toxin RelE of M.tb affected
growth of the E. coli and M.tb. The three RelE toxins of M.tb are involved in
individual antibiotic specific tolerance (Singh et al. 2010).
The YefM antitoxin is highly unstable as it is prone to degradation by Lon, an
ATP-dependent serine protease. YefM is co-expressed with the YoeB toxin and the
resultant complex so formed consists of dimer of YefM and single molecule of YoeB
(Kamada and Hanaoka 2005). Rv3357–Rv3358 of M.tb codes for YefM/YoeB
system.
The HigBA family, HigB toxin (Rv1955) and HigA antitoxin (Rv1956), are part
of the operon comprising of Rv1954A and Rv1957. Rv1957 is found as a SecB-like
chaperone required for antitoxin stabilization. HigB inhibits protein synthesis by
cleaving mRNAs that are being translated in E. coli (Smollett et al. 2009; Fivian-
Hughes and Davis 2010; Christensen-Dalsgaard et al. 2010; Bordes et al. 2011).
The tripartite toxin-antitoxin-chaperone system (TAC) complex is induced during
heat shock, hypoxia, nutrient starvation, and persistence. Within the TAC complex,
the chaperone directly binds to HigA antitoxin and prevents it aggregation or
degradation, thereby aiding in HigA folding and successive interaction with HigB
toxin (Bordes et al. 2011).
M.tbH37Rv also possesses two ParDE systems, and ParDE2 operon has been
investigated recently and was found that toxin MParE2 interacts with GyrB subunit
and inhibits bacterial growth by inhibiting DNA gyrase, thereby blocking DNA
replication (Gupta et al. 2016).
15.3 Types of Toxin-Antitoxin Systems
TA system is characterized by neutralization of toxin by the antitoxin (Fig. 15.1). In

case of a type I TA system, translation of messenger RNA encoding the toxin is
inhibited by binding of a non-coding RNA antitoxin to the mRNA. The protein toxin
in case of type II TA system is inhibited post-translationally through binding of
another protein antitoxin. In case of type III TA systems, a small RNA binds directly
to the toxin protein. Type IV-VI TA systems are also reported but are relatively less
common. Toxin-antitoxin genes, transferred predominantly through horizontal gene
transfer, are mostly associated with pathogenic bacteria and with plasmids confer-
ring antibiotic resistance or virulence (Mine et al. 2009; Van Melderen and Saavedra
De Bast 2009).
15.3.1 Type I TA System
In this type of TA system, antitoxins consist of small regulatory RNAs (sRNA)

comprising of 50–200 nucleotides. A non-coding RNA antitoxin complementarily
binds to the toxin-encoding mRNA resulting in mRNA degradation or inhibition of
toxin translation (Brielle et al. 2016; Brantl and Jahn 2015). Type I toxin and
antitoxins are transcribed from their own promoter, while in other types of TA
systems, they are part of operon with other genes. The translation of the type I
toxin mRNA is inhibited by base pairing with the antitoxin sRNA that prevents
interaction with ribosome resulting in inhibition of translation of toxin mRNA
(Brantl 2012; Fozo et al. 2008). Most of the type I toxins are small hydrophobic
proteins that create pores in the inner membrane leading to breakdown of membrane
potential to stop ATP synthesis, thereby blocking energy-demanding activities such
as protein synthesis (Wen et al. 2014; Lee and Lee 2016). symR/symE module of
E. coli is considered as an example of type I TA system (Kawano et al. 2007).
262 A. Kumar et al.
Fig. 15.1 Types of toxin-antitoxin systems: Toxin proteins inhibit the growth of cells and there are
different mechanisms through which antitoxins neutralize the effect of toxins: (a) Suppression in
translation of toxins by complementary RNA complex formation between toxin and antitoxin
mRNA. (b) Non-functional toxin-antitoxin protein complex neutralizes the effect of toxins on
growth. (c) Non-functional complex formation due to interaction of antitoxin mRNA with toxin
proteins. (d) Interaction of antitoxin with target proteins stops toxin activities. (e) Antitoxin protein
degrades mRNA of toxin resulting in suppression of toxin protein synthesis. (f) Antitoxins facilitate
degradation of toxins by proteinases resulting in rescue of cell growth
15.3.2 Type II TA System
Type II TA systems are most extensively characterized in both prokaryotes and

archaea. In type II TA system, the functional activity of toxin proteins is inhibited
due to interaction between stable toxin and labile antitoxin. Toxin and antitoxin
proteins are expressed simultaneously as both are organized into operons. The
activity of different toxin protein regulates various mechanisms within the cell.
For example, CcdB protein of E. coli (strain K12) inhibit the function of DNA
gyrase by inactivating DNA topoisomerase II, whereas MazF cleaves cellular
mRNAs for the inhibition of protein synthesis (Bernard and Couturier 1992;
Zhang et al. 2003). The TA complex acts as a repressor for TA operon system as
it binds to the palindromic sequence of the promoter region. Due to antitoxin
degradation, the concentration of TA complex reduces thereby leading to production
of more toxins and antitoxin.
15.3.3 Type III TA System
Type III TA system is characterized by direct interaction between a toxic protein and
RNA antitoxin. The RNA gene involved totally neutralizes the effects of the toxic
protein. ToxI-ToxN TA system from a plant pathogen named Erwinia carotovora
subspecies atrosepticum (Pectobacterium atrosepticum) is a perfect example of type
III TA system. The function of toxin protein (ToxN) is directly suppressed by
interaction with antitoxin RNA, forming aToxN-RNA (an RNA antitoxin) complex
(Fineran et al. 2009; Blower et al. 2012).
15.3.4 Type IV TA System
In type IV TA system, there is no direct interaction between toxin and antitoxin proteins.
Antitoxin protein interacts with the target of toxin protein, thereby suppressing the
activity of toxin on its target. The functional aspect of type IV TA system is exemplified
in case of toxin CbeA of E. coli (1303) that prevents polymerization of the cytoskeletal
proteins (MreB and FtsZ) and inhibits cell division. The antitoxin CbeA (YeeU) protein
inhibits by binding directly with the target, namely, MreB and FtsZ toxin, instead of
forming a toxin-antitoxin complex (Masuda et al. 2012).
15.3.5 Type V TA System
In the type V TA system, antitoxin protein retards synthesis of toxin protein by

degrading the mRNA transcribed to code toxin protein. This is exemplified by GhoT
264 A. Kumar et al.
of E. coli (strain K12), a protein that induces persistence and ghost cell formation with
damaged membrane. The antitoxin GhoS is an endoribonuclease and precisely cleaves
the mRNA encoding for membrane-lytic peptide toxin GhoT (Wang et al. 2013).
15.3.6 Type VI TA System
In the type VI TA system, antitoxin protein facilitates the proteolytic degradation of

toxin protein. There is no degradation of toxin protein by proteases in the absence of
antitoxin, and if toxin-antitoxin proteins are together, toxin degradation occurs. In
SocAB TA system in Caulobacter crescentus, SocB protein inhibits DNA elonga-
tion by intercalating with DnaN, whereas antitoxin SocA facilitates degradation of
SocB in the presence of protease ClpXP (Aakre et al. 2013).
15.4 Biological Roles of Toxin-Antitoxin Systems
TA systems regulate bacterial survival in different types of unfavourable conditions.

TA systems are involved in several biological functions such as growth regulation,
physiological changes of the cells, programmed cell death, etc. (Fig. 15.2). A more
detailed importance of the TA systems in various physiological conditions is
described below:
Fig. 15.2 Roles and applications of toxin-antitoxin systems: Toxin-antitoxin systems play impor-
tant roles such as plasmid maintenance, drug resistance/tolerance, biofilm formation, growth
regulation, antiphage activity, genotyping, programmed cell death, etc.
15.4.1 Stress Survival
TA systems act on regulatory machinery that control mechanisms critical for sur-
vival of bacteria (Engelberg-Kulka et al. 2006; Yamaguchi et al. 2011). For example,
TA systems are important for adaptation of M.tb to unfavourable environmental
conditions inside the host and maybe required for triggering a non-replicating state
(Lewis 2007). Mycobacteria often adopt a non-replicative persistent, inactive state,
to avoid unfavourable stress conditions like hypoxia, oxidative stress, nutritional
limitations, acidic pH, etc., within the host macrophages (Wu et al. 2012). There are
88 putative TA systems present in M.tbH37Rv that are conserved in M.tb complex
(MTBC) but are few in other non-pathogenic mycobacteria, indicating a potential
contribution to the pathogenic lifestyle of M.tb (Ramage et al. 2009). In response to
starvation, several toxins within the cells are upregulated leading to inhibition of
translation and selective degradation of mRNA. Contrary to starvation which leads
to generalized upregulation of TA loci, low pH exposure leads to downregulation of
few TA genes (Gupta et al. 2017).
15.4.2 Persistence
In majority of bacteria, there are set of genes that are involved in growth inhibition,
and their overexpression may result in cell death, similar to programmed cell death
of eukaryotes (Wen et al. 2014). It has been reported that TA systems are important
for persister phenotype in E. coli (Tsilibaris et al. 2007). TA systems present in M.tb
may regulate cell division during infection (Warner and Mizrahi 2006). After
infection, M.tb initially grows and then acquires latency, a state of non-replicating
cells in unfavourable conditions that can survive long periods with the potential to
reactivate itself later whenever environment is favourable (Stewart et al. 2003, North
and Jung 2004). In the case of persistence, a subpopulation of non-replicating
bacteria becomes tolerant to antibiotics (Gomez and McKinney 2004). The presence
of TA system in M.tb, as in other bacteria, imparts antibiotic tolerance and is one of
the main reasons why long term of antibiotic therapy is required to cure tuberculosis
(Keren et al. 2004; Ramage et al. 2009). It has been reported that mycobacterial
MazF ribonucleases are involved in the drug tolerance, adaptation in oxidative
stress, and nutrient depletion and virulence (Tiwari et al. 2015). M.tb RelE toxins
are involved in formation of persisters specific to individual antibiotic and involved
in drug-specific tolerance (Singh et al. 2010).
15.4.3 Biofilms
Planktonic bacteria can aggregate and attach themselves on biotic or abiotic surfaces
to form biofilms. Formation of biofilms by pathogens is considered to be one of the
266 A. Kumar et al.
main survival strategies to counter the host defence (Rybtke et al. 2011; Kumar et al.
2017a). TA systems have been shown to be involved in biofilm formation, with
exceptions. It has been shown that type II TA system of E. coli involving MqsR
protein is induced during biofilm formation and deletion of this gene resulted in the
absence of biofilm formation (Kasari et al. 2010). The role of mqsRA TA system in
biofilm formation is associated with motility and as autoinducer-2 quorum sensing
system (Gonzalez Barrios et al. 2006). The yefM-yoeB and relBE TA systems of
Streptococcus pneumonia are involved in the biofilm formation. It has been reported
that mutant strains lacking yefM-yoeB or both yefM-yoeB and relBE show reduc-
tion in biofilm formation (Chan et al. 2018). It has also been shown that deletion of
mutants of mazF and relE gene homologue did not affect biofilm formation in
Streptococcus mutans (Lemos et al. 2005).
15.4.4 Antiphage Activity
The competitive edge of pathogen against the host is driven by the enormous
diversity and multiplicity of TA systems. Bacteria have evolved several defence
mechanisms to protect themselves and survive against the onslaught of phage
infection. Bacteria exhibit a wide array of mechanisms to resist bacteriophages
that include inhibition of adsorption, exclusion of superinfection, cleavage of nucleic
acids of the phages through restriction-modification or CRISPR-Cas systems, and
abortive infection (Stern and Sorek 2012; Seed 2015). The abortive infection
systems trigger premature death of phage-infected bacteria and restrict the phage
to replicate or spread, thereby protecting the uninfected bacterial population within
the niche. Thus, there is a link between abortive infection and TA systems. Several
TA systems have anti-phage activity including Hok-Sok, LsoAB and MazEF,
ToxIN, and AbiEG (Short et al. 2018).
15.4.5 Bacterial Virulence and Pathogenecity
Presence of TA modules in the genome is directly related to the virulence of bacteria

(Georgiades and Raoult 2011), for example, type I toxins are involved in lysis of
host cell. The PepA1 toxin of S. aureus is a pore-forming peptide that causes
bacterial cell death. When there is oxidative stress inside host cell, the PepA1
toxin is typically released from its SprA antitoxin. This is an example of altruistic
behaviour, as the peptide drives erythrocyte lysis, resulting in release of slowly
dividing cells that escape the immune system (Sayed et al. 2012). Deletion of
VapBC homologues in Haemophilus influenzae results in remarkable decrease of
virulence in animal models for otitis media, tissue, etc. (Ren et al. 2012).
15.4.6 Growth Regulation
M.tb encounters different types of unfavourable conditions during infection. To

survive in stressful conditions, bacteria regulate its growth. There are several
proteins that are involved in growth regulation other than TA systems such as
DATIN, IciA, MSMEG_1878 (M.tbRv3241c orthologue in M. smegmatis), etc. M.
tb DATIN and MSMEG_1878 inhibit protein synthesis by interacting with ribosome
(Kumar et al. 2012; Li et al. 2018). M.tb IciA inhibits bacterial growth by inhibiting
the opening of two strands of DNA during replication (Kumar et al. 2009; Kumar
et al. 2017b). Toxin-antitoxin systems are activated during starvation stress. The
RNase toxins, instead of being bactericidal, are usually bacteriostatic in nature.
There is quick arrest of growth in response to starvation or other environmental
stresses which help in their survival, and quicker resumption of growth occurs when
the situation improves (Gerdes 2000; Gerdes et al. 2005).
15.4.7 Programmed Cell Death
Programmed cell death (PCD or apoptosis) is a physiological process and occurs

mainly in multicellular, eukaryotic organisms during the process of embryonic
development or tissue turnover. Dysregulation of PCD results in diseases like
tumour formation, autoimmune diseases, or lysosomal disorders (Hayes 2003).
Bacteria, being unicellular, do undergo PCD. However in natural environment,
bacteria exist as biofilms that represent multicellular colonies and display coordina-
tion as in multicellular organisms. Such immobilized bacteria maintain discrete and
ordered spatial structures within the biofilm niche. There are several genes in
bacteria that are homologues to eukaryotic genes involved in PCD (Koonin and
Aravind 2002), and TA modules of E. coli are either PCD genes or mediators of
reversible growth arrest, which alternatively might allow the cells to enter a dormant
or a semi-dormant state. It has been shown that PCD in bacteria might allow
surviving cells to scavenge nutrients from dead ones and may prevent spread of
bacteriophages (Engelberg-Kulka and Glaser 1999).
15.5 Applications of Toxin-Antitoxin Systems
There is a growing need for new antimicrobial agents due to decrease in the
effectivity of drugs being used currently arising out of increase in multi-drug
resistance. The toxins of TA systems usually target various biological processes
such as replication, transcription, translation, macromolecular synthesis, cell wall
synthesis, phage infection, and cytoskeletal polymerization. The fact that some of
these toxins also overlap with the targets of the antibiotics (Wen et al. 2014) provides
an option to explore TA systems as drug target in bacteria. There are several
antibiotics that act indirectly against the TA systems. The detailed study of the
interaction between the toxin and the antitoxin may help in the formulation of new
268 A. Kumar et al.
drugs. Mapping the precise location of different TA systems in bacterial chromo-

some or plasmid will uncover fundamental insight into their possible applications as
drug targets.
Previously, IS-elements, DR-elements, variable number tandem repeats (VNTR),
and single-nucleotide polymorphisms (SNPs) were used as genetic markers in
housekeeping genes or other genes and were used for genotyping. The type II TA
systems are involved in virulence, persistence, and survival of M.tb inside host
macrophages (Zaychikova et al. 2015). Analyses of 173 sequenced genomes of M.
tb for the genes of type II TA systems show genetic diversity (SNPs) that correlates
with the specific genotype of M.tb strains (Zaychikova et al. 2015). This correlation
between a genotype of particular strain and SNPs in different genes of type II TA
system paved way to consider TA systems as a new biomarker for genotyping of
tuberculosis caused by different strains of M.tb.
15.6 Conclusion
Genome sequencing and its annotation have suggested presence of significant

number of TA systems in microorganisms. The classification of TA system is
based on similarity of primary sequences and on the specificity of interaction
between pair of toxin and antitoxin molecules. Type II system is structurally most
flexible in terms of location of antitoxin gene either upstream or downstream of the
toxin gene or regulation of repression activities that is encoded by different genes.
The various types of TA system may shuffle among themselves in terms of function
and can exhibit ‘mix and match’ phenomenon. This is exemplified by the structural
similarity between GinI solitary toxins with type II HicA toxin. In some cases, toxins
can also evolve from the antitoxin under selective pressure. As in the case of VapD
sequences, which mediate defence against phages, TA system points to a common
origin with CRISPR-cas system. There is consensus that the evolution of TA system
in bacteria enabled these unicellular organisms into robust molecular machines that
could withstand the onslaught of environment and various stress. The importance of
TA system is also underlined by the fact that while many pathogenic and obligatory
intracellular pathogens opted for reductive evolution in genome size and shed off the
extra burden of many genes, yet they retained the genes associated with TA system.
The distribution of TA system varies even among strains of the same species of
bacteria. TA systems are highly mobile in nature and moves between genomes
through horizontal gene transfer. The presence of TA genes within the transposons
makes them refractory to gene efflux and stabilizes the TA system within the cells.
The additive property of TA genes within the transposons facilitates increased
stability and exclusion of foreign DNA. Although the activity of TA system may
be subdued by host cell machinery, it may rescue its activity similar to the restriction
modification system. TA systems are preferentially associated with genomic islands
or plasmids which serve as a mechanism to maintain their structural integrity during
stress and survival through post-segregational killing. M.tb possesses a huge number
of TA systems that are usually located within distinct genomic islands and trigger
decrease in metabolic activity. In case the TA genes are integrated within the core
genome, these accumulate mutation that may lead to loss of addictive property or
deletion of TA system. In case of E. coli, type I hok-sok system are inactivated by IS
sequences, gene rearrangements, and point mutations. TA system can evolve
through integration with the host regulatory machinery and may replace the antitoxin
molecules with signal transduction molecules.
Toxins have many cellular functions including inhibition of protein synthesis,
DNA replication, and synthesis of cell wall in response to unfavourable conditions.
Some toxins in TA system act as ribonucleases, while some other toxins act as
gyrase inhibitors and kinases. The TA genes express in different stressed conditions
such as nutrient deficiency, antibiotic treatment, bacteriophage infection, host
immune responses, oxidative stress, and high temperature. They are involved in
persistence, slow cell growth, cell cycle arrest, or cell death. Proteins involved in TA
systems may act as important targets for drug development that may help in
reduction of treatment duration of tuberculosis and other infectious diseases. In
spite of the diversity in structure of TA system, the function of the TA system is
tightly regulated by other cellular networks such that the prolific activity of TA
system is activated only as a response to cellular physiology and the toxins are
unleashed for minimal activity. The activity of TA locus that regulate activation of
signaling pathways involved in persister cell formation in biofilms are modules
acting as effectors of persister cell formation. These are usually deeply integrated
into cellular signaling pathways that tightly control their activation and use their
characteristic auto-regulatory features to tune the induction, duration, and intensity
of the phenotypic switch into dormancy.
It is speculated that our current understanding of the toxin-antitoxin system is still
redundant and a deeper understanding of the mechanistic significance of TA system
will enable us to unravel the mysteries of how these unicellular organisms could
dominate the environment. It is envisaged that unlocking the mechanism of TA
system could allow us to shape better strategies for overcoming the harmful effects
of TA system in clinical pathogenesis and in dealing with microbial drug tolerance.
Funding This project has been funded by “UGC-BSR Research Start-Up-Grant project
No. F. 30-487/2019(BSR) sanctioned to Ashutosh Kumar”
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Nucleotide Excision Repair Pathway
in Mycobacteria 16
Manoj Thakur and K. Muniyappa
Abstract
Nucleotide excision repair (henceforth abbreviated as NER) plays a pivotal role in
all organisms to protect their genetic material against radiations, toxic chemicals,
and normal by-products of cellular metabolism. In humans, defects in excision
repair causes inherited diseases, and NER-related human diseases are associated
with cancer and aging. Much of our current understanding of NER has emerged
from experimental evidence in model systems including Escherichia coli, yeast,
and mammalian cells. Considering the importance of NER in the maintenance of
genome integrity, it is surprising that only a few studies have investigated NER in
mycobacteria. Here we provide a brief overview of the mechanism of the DNA
repair in mycobacteria. A detailed understanding of structure-function relation-
ship of DNA repair proteins in tubercle bacillus could facilitate the identification
and development of novel therapeutic targets for tuberculosis therapy.
Keywords
DNA damage · DNA helicase · Mycobacterium tuberculosis UvrA · UvrB ·
UvrC · UvrD · Nucleotide excision repair · SOS response
Abbreviations
ATP adenosine triphosphate

BCG Bacillus Calmette–Guerin
BER base excision repair;
CFU colony-forming units
M. Thakur · K. Muniyappa (*)

Department of Biochemistry, Indian Institute of Science, Bangalore, India
e-mail: kmbc@iisc.ac.in

https://doi.org/10.1007/978-981-32-9413-4_16
276 M. Thakur and K. Muniyappa
DSBs double-strand breaks

dsDNA double-stranded DNA
EcRecA Escherichia coli RecA
EcUvrA Escherichia coli UvrA
EcUvrB Escherichia coli UvrB
HR homologous recombination
iNOS inducible nitric oxide synthase
MMR mismatch repair
MtRecA Mycobacterium tuberculosis RecA
MtUvrB Mycobacterium tuberculosis UvrB
NER nucleotide excision repair
NHEJ non-homologous end joining
RNI reactive nitrogen intermediates
ROI reactive oxygen intermediates
ssDNA single-stranded DNA
TCR transcription-coupled repair
UV-light ultraviolet-light
XDR extensively drug resistant
16.1 Overview of DNA Damage Response Mechanisms
Numerous studies have established that the genomic DNA undergoes damage
inflicted by radiation, chemicals, environmental agents, and internal metabolic
(endogenous) processes (Hoeijmakers 2001; Lindahl and Wood 1999). These pro-
duce nucleotide modifications such as bulky adducts, single- and double-strand
breaks, inter- or intra-strand cross-links, DNA-protein cross-links, apurinic or
apyrimidinic base-free sites, misincorporation of nucleotide or ribonucleotide by
DNA polymerases, and DNA replication slippage (Vaisman et al. 2013; Dexheimer
2012; Lindahl and Wood 1999; Van Houten and Sancar 1987). If these lesions
remain unrepaired, they lead to genomic instability and various diseases like cancer,
neurological disorders, immunodeficiency, premature aging, and Fanconi anaemia in
humans (Iyama and Wilson 2013). Therefore, maintenance of genome stability
under normal and stressed conditions becomes an important task to the cellular
repair machinery.
To circumvent the harmful or deleterious effects of myriad types of DNA
damage, cells have evolved specialized types of DNA repair systems. These can
be classified based on the protein components and mechanism(s) involved as well as
different types of DNA lesions being processed. The different DNA damage repair
pathways include direct reversal of damaged base, base excision repair (BER),
mismatch repair (MMR), nucleotide excision repair (NER), and single- and
double-strand breaks (DSBs) repair (Fig. 16.1). The repair of both single- and
16 Nucleotide Excision Repair Pathway in Mycobacteria 277
Fig. 16.1 Overview of DNA

damage and repair
mechanisms. The figure
illustrates common types of
DNA lesions and the specific
mechanism to repair each type
of lesion (this Figure is cited
from Hakem (2008).
(Reproduced with the
permission of EMBO J)
double-strand breaks involves homologous recombination (HR) and

non-homologous DNA end-joining (NHEJ) pathways (Kuzminov 2001; Cannan
and Pederson 2016; Friedberg 2016; Yi and He 2013; Hakem 2008). The repair of
damaged bases involves three mechanistically distinct processes: (1) photolyase
catalysed reversal of UV light-induced photolesions; (2) O6-alkylguanine-DNA
alkyltransferases (AGTs) catalysed reversal of various O-alkylated DNA damage;
and (3) the AlkB family dioxygenases mediated reversal of N-alkylated base adducts
(Yi and He 2013). The photolyase enzyme reactivates DNA by direct repair of T<>T
lesions, and BER is used to repair single-stranded breaks and to correct the damaged
bases resulting from spontaneous DNA deamination, hydroxylation of bases, or
exposure of DNA to alkylating agents. On the other hand, MMR recognizes and
repairs erroneous insertions, deletions, and misincorporation of bases arising from
DNA replication, and base deamination. This system recognizes the presence of
hemimethylated dGATC sequences that provides molecular basis to distinguish
between the newly synthesized daughter strand and parental DNA strand (Radman
and Wagner 1986). This hemi-methylated dGATC site 50 or 30 to the mismatch then
cleaved by the concerted action of MutS, MutL, and MutH in an ATP-dependent
manner (Acharya et al. 2003). ExoI or ExoX (30 ! 50 exonuclease), or ExoVII or
RecJ (50 ! 30 exonuclease) excise the cleaved strand up to and slightly past the
mismatch. The resulting single-stranded gap is repaired by DNA synthesis using
intact complimentary strand as a template and ligated by DNA polymerase III or
DNA ligase (Li 2008).
The double-stranded breaks are repaired via either HR or NHEJ, but what
determines the pathway preference remains elusive (Lieber 2010). In eubacteria,
HR comprises of four stages: (1) when DSBs occur, a process called DNA end
resection is activated to generate 30 single-stranded overhangs; (2) RecA
polymerizes onto 30 -single-stranded DNA (ssDNA) to form a nucleoprotein fila-
ment; (3) RecA nucleoprotein filament initiates search for homology, pairing, and
strand exchange; and (4) finally, the extension of heteroduplex DNA by RuvAB
followed by resolution of the Holliday junction by RuvC produces crossover and
non-crossover products (Cox 2007; Kowalczykowski et al. 1994). The bacterial cells
also depend on NHEJ during pre-replicative stage when only single copy of the
genome is available. Genetic and biochemical evidences have also shown that
mycobacteria possess NHEJ pathway that depends on DNA ligase (LigD) and Ku
protein (Matthews and Simmons 2014; Shuman and Glickman 2007). Although the
biochemical mechanisms of all these pathways have been extensively studied in
considerable depth in bacteria, how these repair events are modulated within the
nucleoid is not fully understood.
16.2 DNA Damage and SOS Response
The induction of the SOS response in bacteria is a global response to DNA damage
in which cell division is arrested and DNA repair is activated by the expression of
DNA damage repair genes (Adikesavan et al. 2011; Schlacher and Goodman 2007;
Radman 1975). In Escherichia coli, the SOS response involves two key proteins:
LexA (a repressor) and RecA (an inducer) as shown in Fig. 16.2 (Radman 1975).
Under normal growth conditions, LexA dimer binds to a 20-base pair consensus
palindromic DNA sequence (also known as the SOS box) and represses the tran-
scription of a regulon (comprising of more than 50 genes, including lexA and recA)
Fig. 16.2 DNA damage and SOS response. Initially the LexA repressor binds to SOS box, present
upstream of SOS genes. Under DNA-damaging conditions, the single-stranded DNA (ssDNA) that
originates from double-strand breaks (DSBs) repair pathway binds RecA proteins leading to the
formation of RecA-nucleoprotein filaments. These filaments induce the autocatalytic cleavage of
LexA allowing derepression of SOS genes. (Figure adapted from Butala et al. 2009)
(Adikesavan et al. 2011). Upon DNA damage, RecA forms a nucleoprotein filament
on ssDNA (Cox 2007; Kowalczykowski et al. 1994). The RecA nucleoprotein
filament facilitates the self-cleavage of LexA, leading to the derepression of SOS
regulon genes (Kowalczykowski et al. 1994). The uvr genes are among the first to be
induced, followed by lexA and recA genes, while genes encoding the low fidelity,
error-prone repair DNA polymerases Pol II (polB), Pol IV (dinB), and Pol V (umuC
and umuD) are induced under extensive and persistent DNA-damaging conditions
(Butala et al. 2009; Smith and Sharma 1987; Smith et al. 1987). These DNA
polymerases allow DNA replication over the DNA lesions (translesion DNA syn-
thesis) that block the primary replicative DNA polymerase Pol III and leads to
mutations in the newly replicated and repaired regions. The uvrA, uvrB, and ydjQ
(cho) genes are SOS-induced genes, whereas uvrC gene is not regulated by SOS
response (Janion 2001, 2008; Simmons et al. 2008; Bohr 1991; Calsou and Salles
1985; Youngs et al. 1974). Although the mechanism of UvrABC mediated repair of
UV-induced photoproducts have been studied extensively under in vitro conditions,
how this system is organized in vivo is unclear. Some estimates indicate that at least
17 protein components are recruited to the inner membrane of E. coli after UV
irradiation. The colocalization of UV-induced 6–4 photoproducts and NER proteins
to the inner membrane following UV-irradiation suggests that a part of the repair
process may associate with the inner membrane (Lin et al. 1997).
The Mycobacterium tuberculosis recA gene encodes an 85 kDa precursor, which
is processed to generate a 38 kDa functionally active RecA and 47 kDa intein (Davis
et al. 1991; Kumar et al. 1996). The X-ray structure of M. tuberculosis RecA
(MtRecA) is very similar to that of E. coli RecA (EcRecA) (Datta et al. 2000).
The transcriptional regulation of M. tuberculosis recA differs from that of E. coli
recA: M. tuberculosis recA is regulated by two promoters (Movahedzadeh et al.
1997b). One of the promoters is regulated by LexA repressor, while the second
promoter is DNA damage-inducible, but independently of both LexA and RecA
(Davis et al. 2002b). The M. tuberculosis LexA-binding site (SOS box) is similar to
that of Bacillus subtilis (Cheo box) (Movahedzadeh et al. 1997a). Further
investigations revealed the existence of several LexA-regulated genes in
M. tuberculosis (Davis et al. 2002a). The apparent lack of correlation between the
expression of DNA damage-inducible genes and the LexA repressor in
M. tuberculosis supports an alternative mechanism of LexA-independent DNA
damage induction (Brooks et al. 2001). Despite this, M. tuberculosis genome
possesses the canonical core of the SOS system, except polB and umuD (Cole
1998; Mizrahi and Andersen 1998). The global gene expression following DNA
damage in both wild-type and recA strain of M. tuberculosis revealed that most of
the genes induced by DNA damage do not have any known roles in DNA repair.
However, another set of the inducible genes with a known or predicted function in
DNA repair was found to be independent of recA (Rand et al. 2003). This study also
revealed yet another group of genes, lexA itself, ruvA, ruvB, and dnaE2 whose
expression was strictly dependent on recA for induction and group of genes whose
expression appear to be regulated by both recA-dependent and recA-independent
mechanisms, e.g. recA itself, radA, ruvC, and ssb.
16.3 Discovery of Nucleotide Excision Repair Genes
Briefly, the survival of E. coli K-12 cells following UV radiation led to the discovery
of NER genes (Boyce and Howard-Flanders 1964; Setlow and Carrier 1964;
Howard-Flanders et al. 1962a). In early studies, thymine dimers were found to be
the main photoproducts after UV irradiation (Setlow and Setlow 1962, 1963; Setlow
et al. 1963). The identification of repair-deficient mutants was based on contra-
selection of irradiated T1 bacteriophage particles (Howard-Flanders and Theriot
1962). These studies identified three loci in E. coli K-12 that control the excision
of pyrimidine dimers and these genes were designated as uvrA, uvrB, and uvrC
(Setlow and Carrier 1964; Howard-Flanders et al. 1962a, b, 1966).
16.4 Overview of Nucleotide Excision Repair Pathway
Studies over the past four decades have identified distinct DNA repair pathways and
molecular mechanisms underlying the myriad types of biochemical reactions of various
repair systems. Among various forms of genomic DNA damage, “bulky” DNA lesions
that cause helical distortion in the double helix can lead to replication fork arrest, and
mutations (Reardon and Sancar 2005). The bulky chemical adducts and interstrand
cross-links are the outcomes of UV irradiation and various other mutagens (Kisker et al.
2013; Truglio et al. 2006; Jeggo et al. 2015). In all three domains of life, various proteins
that belong to distinct DNA repair pathways recognize these DNA alterations and then
either repair them by direct reversal (photoreactivation system) or target them for
removal by a series of DNA repair systems (Reardon and Sancar 2005; Kisker et al.
2013; Truglio et al. 2006; Jeggo et al. 2015). One of the most studied and best
understood DNA repair machinery in eubacteria and archaebacteria is the NER pathway
(Jeggo et al. 2015; Kisker et al. 2013; Truglio et al. 2006; Reardon and Sancar 2005;
Ogrunc et al. 1998; Grogan 2000). In these two domains, the NER pathway is dedicated
to the removal of a variety of chemically and structurally diverse DNA lesions,
including UV-induced photoproducts (cyclobutane dimers, 6–4 photoproducts, and
thymine glycol), bulky adducts, apurinic or apyrimidinic (AP) sites, and cross-links,
albeit with variable efficiencies (Goosen 2010; Truglio et al. 2006; Reardon and Sancar
2005; Alekseyev et al. 2004; Selby and Sancar 1991; Snowden et al. 1990; Lin and
Sancar 1989; Van Houten et al. 1988; Van Houten and Sancar 1987; Thomas et al.
1986; Sancar et al. 1985; Seeberg et al. 1983). In addition to the structural distortions in
the DNA, UvrABC can also repair cross-links between protein and DNA as well as the
damages caused by reactive oxygen and nitrogen species (Salem et al. 2009; Imoto et al.
2008; Minko et al. 2002, 2005; Nakano et al. 2005).
In all three domains of life, NER comprises of discrete steps: DNA scanning,
damage detection, damage verification, incision or removal of damage, DNA syn-
thesis, and ligation as depicted in Fig. 16.3 (Hu et al. 2017; Kisker et al. 2013;
ä
Fig. 16.3 (continued) is required for the removal of incised lesion containing small oligonucleo-
tide. Repair is then completed by the action of DNA polymerase I and DNA ligase which
synthesizes the new strand using intact complementary strand as a template and ligates the nick,
respectively. (Figure adapted from Kisker et al. 2013)
Fig. 16.3 Schematic representation of the prokaryotic NER pathways. Genome is scanned by the
heterotetrameric UvrA2–UvrB2 complex in search for damaged nucleotides in an ATP-dependent
manner. After the damage detection by UvrA component, pathway proceeds with damage verifica-
tion by UvrB followed by 30 and 50 incisions catalysed through UvrC. The helicase activity of UvrD
Truglio et al. 2006; Ogrunc et al. 1998;). In prokaryotes, NER can be initiated by two
major pathways often referred to as global genome repair (GGR) and transcription-
coupled repair (TCR). GGR can take place at any region of the genome and is
generally initiated by the direct binding of UvrAB complex to the DNA under
DNA-damaging conditions or during the SOS response (Simmons et al. 2008;
Janion 2001, 2008; Crowley et al. 2006; Hanna et al. 2001). The transcription
coupled NER is initiated by stalled RNA polymerase on the template strand with
the help of TCR specific factor, Mfd (Pani and Nudler 2017; Van Houten and Kad
2014). The TCR not only leads to repair of lesions on the DNA but also
accomplishes efficient and correct transfer of information from genes to RNA.
Although the first step of initiating NER is different in GGR and TCR, both the
processes require the same molecular protein complexes to complete the excision
repair (Van Houten and Kad 2014). Specifically, UvrAB complex bound to ATP
scans the genome for lesions. Once a lesion is detected by UvrA, the lesion
containing strand is transferred to the UvrB for damage verification and UvrA is
released from the process (Pakotiprapha et al. 2012; Jaciuk et al. 2011; Kad et al.
2010; Malta et al. 2007; Truglio et al. 2006). The UvrB subunit binds specifically to
the lesion containing DNA strand by inserting its beta-hairpin domain between the
two strands of the DNA for verification of the lesion. UvrC binds to this tight UvrB-
DNA preincision complex and mediates the incisions on both 30 and 50 sides of the
lesion on the single-stranded DNA (Pu et al. 1989). Many bacterial species such as
E. coli, Mycobacterium tuberculosis, Salmonella typhimurium, Salmonella
enteritidis, Klebsiella pneumoniae, and Clostridium acetobutylicum harbour another
protein called YdjQ (renamed as Cho) which incises only at the 30 side of the lesion.
It was found that Cho cleaves the poor DNA substrates of UvrC very efficiently and
thus further increases the substrate range of NER (Moolenaar et al. 2002). Subse-
quently, UvrD (DNA helicase II) removes the excised lesion containing the oligo-
nucleotide by its helicase action and DNA polymerase I fills the gap using intact
complimentary strand as a template (Brosh 2014; Lee and Yang 2006; Runyon et al.
1990; Caron et al. 1985). Finally, DNA ligase seals the nick; thus genome integrity is
restored (Paul-Konietzko et al. 2015; Orren et al. 1992; Van Houten et al. 1988;
Husain et al. 1985).
Much of the available knowledge regarding NER has come through
investigations in model organisms such as E. coli, yeast, and cultured mammalian
cells. A detailed discussion on the mechanistic aspects of NER is beyond the scope
of this review; however, the reader is referred to excellent reviews that deal with the
mechanistic aspects of NER (Hu et al. 2017; Pani and Nudler 2017; Kisker et al.
2013; Van Houten et al. 2005). While the NER pathway has been studied
extensively over the years, the comparison between E. coli and mycobacteria is
important for identifying possible genetic and mechanistic differences that might
contribute to the observed differences. This review focuses on the function of NER
in mycobacteria and on the role of this pathway in the cellular response to
UV-induced DNA damage.
16.5 DNA Repair in Mycobacterium tuberculosis
The macrophages mount an effective host defence by recognizing, engulfing, and

killing M. tuberculosis (Levitte et al. 2016). The recruitment of macrophages is
through Toll-like receptor (TLR)-mediated signalling that is triggered by pathogen-
activated molecular patterns (PAMPs) present on bacterial surface (Fig. 16.4). The
infected macrophages migrate across the lung epithelium and then transport the
Fig. 16.4 Simplified diagram of the phagocytosis and degradation of a mycobacterial cell.
Phagocytosis is triggered by the binding of Toll-like receptors to the PAMPs on the cell surface
of mycobacteria. After the particle is taken up by the phagocyte, mycobacterial DNA is exposed to
the deleterious effect of ROI generated by the action of phagocyte oxidase (NOX2/gp91Phox). Upon
immunological activation with IFNγ, the phagosome matures and fuses with lysosomes leading to
the production of RNI from iNOS (inducible nitric oxide synthase 2) and ROI from NOX2. These
species react with a wide range of molecules, including nucleic acids, proteins, lipids, and
carbohydrates. If the damaged DNA of M. tuberculosis left unrepaired, the mycobacterial cells
inside the macrophages will undergo apoptosis inside phagolysosomes, and subsequently the cell
debris will be secreted out by the phagocyte. (Figure adapted from Ehrt and Schnappinger 2009)
bacteria to other tissues (Cambier et al. 2014a). Subsequently, new macrophage

recruitment takes place and the original infected macrophages form granuloma, an
aggregation of differentiated macrophages, and other immune cells. In these
granulomas the infection expands by spreading bacteria to newly arrived
macrophages (Cambier et al. 2014b). The experimental evidences suggest a link
between the association of Toll-like receptor signalling, reactive oxygen
intermediates (ROI), and reactive nitrogen intermediates (RNI) (Marcato et al.
2008; Ryan et al. 2004). Such signalling cascades lead to the activation of
macrophages as an innate immune response to kill M. tuberculosis. Studies suggest
that both ROI and RNI can permeabilize the cell wall/membrane of the pathogen
(Fig. 16.4) (Schlosser-Silverman et al. 2000; Fang 1997). Most common damages
that occur in DNA are the base modifications, generation of abasic sites, and strand
breaks in response to RNI and ROI (Wink et al. 1991). Specifically, ROS produces
single pyrimidine and purine base lesions, intrastrand cross-links, purine 50 ,8-
cyclonucleosides, DNA-protein adducts, and interstrand cross-links (Cadet and
Wagner 2013). Therefore, inability to rectify these damages in the DNA inside the
macrophages can compromise its survival and ability to cause pathogenesis. Several
studies suggest that M. tuberculosis is well equipped to counteract the harmful
effects of ROS, RNI, and low pH (<3.5) generated by the host immune system
(Ehrt and Schnappinger 2009). But how the genome integrity of M. tuberculosis is
maintained under these conditions continues to be an important area of research. To
this end, genome-based studies in M. tuberculosis showed the involvement of
various DNA repair proteins and nucleoid-associated proteins (NAPs) to circumvent
such DNA damaging conditions (Philipp et al. 1998a, b; Bergh and Cole 1998;
Brosch et al. 1998).
M. tuberculosis harbours all the vital genes of NER, base excision repair (BER),
HR, NHEJ, SOS repair, and mutagenesis, as deduced from bioinformatics and
genome-based studies, but the function of these proteins and enzymes is not fully
understood. Moreover, studies on DNA repair components is important considering
that M. tuberculosis lacks functional mismatch repair pathway (MMR) (Cole 1998).
In contrast to Helicobacter pylori, in which a correlation exists between the absence
of MMR and high level of genetic diversity, M. tuberculosis genomes are very stable
(Kang and Blaser 2006). This observation suggests that other DNA repair
mechanisms could be very efficient in this pathogen. Recently, NucS-dependent
DNA repair system that mimics the MutS/MutL-based MMR has been identified in
Mycobacterium smegmatis (Castaneda-Garcia, et al. 2017). Thus, characterization of
M. tuberculosis DNA repair pathways in vivo and in vitro may contribute to the
understanding of the generation of clonal populations of M. tuberculosis (Sreevatsan
et al. 1997).
The adaptive nature of M. tuberculosis is significant in the clinical context
because of its ability to supress central metabolism, halt DNA replication, and
enter into a stage of dormancy rendering itself extremely resistant to host defence
and drug treatments (Gengenbacher and Kaufmann 2012). Thus, the physiological
changes that take place during the shift from dormancy to reactivation of the tubercle
bacillus should be investigated in detail (Gopinath et al. 2015; Ehrt et al. 2015;
Gorna et al. 2010). To this end, Affymetrix GeneChip System Microarray (based on
a specific oligonucleotide array format) revealed that almost half of the DNA repair
genes are continuously expressed during log phase of growth, indicating that the
bacteria continuously counteract the constant exposure to DNA-damaging
conditions (Fu and Fu-Liu 2007). Additionally, transposon site hybridization
(TraSH) technique identified the genes, Rv2554c, dut, ligA, polA, and adnB and
those encoding the NER-related proteins UvrD1, UvrD2, and UvrC, which are
essential for optimal growth by M. tuberculosis (Sassetti et al. 2003). The uvrD2
and ligA genes were also found to be essential for the survival of M. smegmatis
(Sinha et al. 2008; Korycka-Machala et al. 2006). In another study, every nonessen-
tial gene of M. tuberculosis was disrupted and their effect on the growth rate and
virulence was determined in mice models. This study also revealed a set of DNA
repair proteins required for survival in mice, apart from a variety of proteins from
different pathways. These include three BER proteins (Ung, Nfo/End, and XthA),
MazG, and RecN (Sassetti and Rubin 2003). The proteome profiling of
M. tuberculosis also indicated the role of DNA repair genes during dormancy and
reactivation (Gopinath et al. 2015). Altogether these studies suggest that most of the
M. tuberculosis DNA repair genes are nonessential for survival in the broth, disrup-
tion of these genes lead to attenuation of virulence in mice. A possible link between
survival and spatial-temporal regulation of DNA repair proteins has been established
by the isolation of M. tuberculosis W-Beijing strains. These strains belong to a drug
resistance family which could be divided into several branches based on unique
alterations in three putative antimutator genes: mutT4, mutT2, and ogt (Dos Vultos
et al. 2008; Nouvel et al. 2007; Ebrahimi-Rad et al. 2003).
M. tuberculosis infection is transmitted by aerosol droplets containing bacteria,
coughed out by individuals with active disease, into the lower lungs of a new host.
Various studies show that M. tuberculosis viability in the aerosols decline to 55%,
10%, and almost zero after 5, 30, and 60 min, respectively (Lever et al. 2000;
Loudon et al. 1969). Studies based on artificially generated droplets carrying BCG
pointed out that UV radiation and desiccation sterilize mycobacterial contaminated
air (Peccia and Hernandez 2004; Ko et al. 2002; Riley et al. 1976). Nevertheless,
from these studies it is evident that the viability of mycobacteria drastically reduces
in the aerosols. It is known that intracellular dehydration (desiccation) leads to DSBs
in DNA, whereas UV exposure leads to the generation of cyclobutane pyrimidine
dimers and pyrimidine (6–4) pyrimidine photoproducts, in which two adjacent
pyrimidines are covalently linked to each other. In mycobacteria, the HR and
NHEJ pathways are involved in the repair of DSBs. Interestingly, M. smegmatis
mutant strains lacking RecA and Ku and/or LigD showed that neither of these
proteins are essential for its growth in vitro (Korycka-Machala et al. 2006). Despite
this, both processes are required for survival under desiccation in the broth culture
(Pitcher et al. 2007). Similar studies were also performed by generating mutants that
influence NER in M. smegmatis and M. tuberculosis. Mutations in polA, uvrB, and
uvrD1 of M. smegmatis and uvrB in M. tuberculosis lead to increased susceptibility
to UV radiation in vitro (Guthlein et al. 2009; Darwin and Nathan 2005; Gordhan
et al. 1996). The deletion of both uvrB and uvrD1 in M. smegmatis showed additive
effect on survival in response to UV radiation indicating that one of the proteins is

involved in another DNA repair pathway. The microarray analysis of
M. tuberculosis genes induced under UV treatment in broth cultures indicated that
the levels of expression of HR and NER gene products increase significantly. These
proteins include UvrA, UvrB, UvrD2, PolA, RecA, RuvC, RuvA, LexA, RecX, and
AdnA (Boshoff et al. 2003, 2004). These studies also highlighted a role for several
other proteins like Ogt, Ada/AlkA, DinF, Lhr, DinX, and DnaE2 in DNA repair.
Besides this, neither the relationship between HR and NER proteins nor the effect of
double mutations in NER and HR proteins is completely understood in
mycobacteria.
16.6 DNA Repair Pathways Are Active During Macrophage

Infection
The genome of M. tuberculosis is exposed to DNA-damaging conditions inside the

granulomas (Adams et al. 1997; Moller et al. 2001). Mice with a non-functional
allele of gp91phox subunit of phagocyte oxidase cytochrome b and iNOS KO mice
lacking an inducible nitric oxide synthase (iNOS) gene fail to produce RNS. Both
these strains of mice experience exaggerated growth of mycobacterial infection
(Adams et al. 1997; MacMicking et al. 1997; Chan et al. 1995). The microarray
analysis revealed that the expression levels of genes involved in NER and BER are
upregulated in the activated macrophages. Apart from these, M. tuberculosis ada/
alkA transcript levels increase significantly in human or murine macrophages as well
as in response to H2O2 in broth culture (Fontan et al. 2008; Schnappinger et al.
2003). Moreover, deletion of ada/alkA gene in M. tuberculosis leads to increased
susceptibility to RNIs, but survival was found to be unaffected (Durbach et al. 2003).
Similarly, deletion of uvrB gene in M. smegmatis and M. tuberculosis also lead to
increased susceptibility to RNIs in broth cultures (Guthlein et al. 2009; Darwin and
Nathan 2005).
The characterization of human granuloma is an active area of research (Tsai et al.
2006). Human granulomas are generally comprised of a central core with CD68+
epithelioid cells, surrounded by macrophages, T lymphocytes (predominantly
CD4+), and multi-nucleate giant Langerhans cells. Another characteristic feature
of human granulomas, which is different from murine granulomas, is the presence of
central acellular eosinophilic region formed due to necrosis and apoptosis of cells.
Because this central region does not possess any vascularization, the environment
surrounding these cells becomes hypoxic (Aly et al. 2006). Proteome profiling,
microarray analysis, and molecular genetic studies have shown that
M. tuberculosis survives within granuloma by varying the expression levels of
DNA repair genes, the expression of some of these genes were either upregulated
or downregulated significantly under hypoxic conditions (Gorna et al. 2010; Kim
et al. 2008; Saunders and Britton 2007; Bacon et al. 2004; Voskuil et al. 2004;
Muttucumaru et al. 2004; Smeulders et al. 1999). In vitro studies show that
M. smegmatis strains deleted for ung and uvrB genes display increased sensitivity
to hypoxia (Kurthkoti et al. 2008; Kurthkoti and Varshney 2012). Studies with
artificial granulomas of mice based on hollow-fibre model revealed that uvrA and
recF were upregulated during dormancy stages (Karakousis et al. 2004). Mice
exposed to aerosolized virulent strain of M. tuberculosis develop persistent infection
and become chronic after 45–60 days post-infection. Transcriptional profiling during
chronic and reactivation phases of murine tuberculosis using in vivo microarray
analysis (IVMA) shows that even after 28 days of aerosol infection, CFU counts
were not changed, although the bacilli were metabolically active with a 50% active
transcriptome. A total of 137 genes were significantly regulated in mid-chronic
tuberculosis (45 and 60 days) compared to an early stage (14 days) where the
expression of genes involved in lipid and carbohydrate metabolism was significantly
enriched. Additional genes, including the virulence regulator virS and the DNA
repair protein ung, were upregulated during the reactivation stage, and hupB and
recO were downregulated, indicating their possible roles in mycobacterial resur-
gence (Talaat et al. 2007). Altogether, these findings along with genome-wide
expression analyses from human lung samples indicate that, while residing in
granulomatous tissue, M. tuberculosis undergoes several changes in the expression
of DNA repair genes.
The reactivation of infection begins with the disintegration of granulomas
because of the failure of macrophages or T cells and with the decline in the host’s
immunity. Although little is known about reactivation of infection, various studies
based on the effect of gene and/or protein expression levels or their activity on
survival implicate a role for mycobacterial DNA repair genes. In vivo studies with
immunocompromised BALB/C mice suggested upregulation in the expression
levels of M. tuberculosis genes ogt, dinG, ung, and recA during reactivation. Of
note, even though the gene expression profiles of all isolates were found to be
similar, the profiles of upregulated DNA repair genes were different in granuloma,
pericavity, and distant lung tissue (Talaat et al. 2007). The observed differences
could be due to dissimilar physiology of cells; thus additional studies are needed to
understand the complexity and reactivation of infection mounted by this respiratory
pathogen.
16.7 Mycobacterium tuberculosis and Chemotherapy
The continuous exposure to the antibiotics leads to the emergence of drug resistance
strains in M. tuberculosis (Karunakaran and Davies 2000; Martinez and Baquero
2000). Drug resistance strains can be divided into two subgroups: MDR (multidrug-
resistant) and extensively drug-resistant (XDR) strains. These notations are based on
the fact that MDR strains are resistant to two front-line drugs, isoniazid and
rifampicin, whereas XDR strains are resistant to isoniazid and rifampicin including
fluoroquinolones and second-line drugs (capreomycin, kanamycin, or amikacin). In
general, antibiotic resistance is thought to arise from horizontal gene transfer carried
by plasmids or transposons and random mutations in the chromosomal genes
(Davies 1997). The genome-wide studies have indicated that the occurrence of
drug-resistant strains is due to the generation of spontaneous mutations or gene

inactivation (Cubillos-Ruiz et al. 2008; Filliol et al. 2006; Tang et al. 2005; Arnold
et al. 2005). For instance, resistance towards anti-tubercular agents has been linked
to SNPs in drug target genes, including rpoB, katG, and uvrB (Eldholm et al. 2014;
Ramaswamy et al. 2003; Zaczek et al. 2009). Like all other organisms, DNA repair
genes directly influence the mutation frequency in mycobacteria. For example,
deletion of ung, uvrB, uvrD, fpg, and udgB in M. smegmatis and ada/alkA together
with ogt in M. tuberculosis leads to increase in mutation rates (Dos Vultos et al.
2009). In view of these results, it was speculated that the use of antibiotics in
combination with new drugs targeting the DNA repair genes might increase the
effectiveness of chemotherapy. For example, it was found that recA-deficient mutant
of BCG had increased susceptibility to metronidazole in vitro and an ogt mutant of
BCG had increased susceptibility to isoniazid (Wiid et al. 2002; Sander et al. 2001).
However, with the use of each drug, it would be important to ascertain that targeting
DNA repair proteins do not result in an increased mutation rate that could lead to the
emergence of drug-resistant strains (Gorna et al. 2010).
16.8 Why Study NER in Mycobacterium tuberculosis?
Although much is known about NER in E. coli, gaps remain in our understanding of
this pathway in other bacteria. Several studies have provided experimental evidences
that the damage search/recognition complex is comprised of two each of UvrA and
UvrB subunits, but exactly how these subunits distinguish damaged from undam-
aged DNA remains unknown. Among many unanswered questions, the oligomeric
status of UvrB and the nature of the preincision complex are unclear. Do two UvrB
subunits bind at the site of a lesion? If UvrB exists as a dimer in solution, then what is
the mechanism underlying the formation of UvrA2B2 complex as seen in
Geobacillus stearothermophilus crystal structure? In this structure, the two UvrB
subunits are located 145 Å apart from each other and positioned 80 Å away from
the lesion (Pakotiprapha et al. 2012). This raises another question: which UvrB
subunit will remain bound to the lesion during the successive steps of NER? Further,
how UvrA dimer specifically transfers damaged strand to UvrB? How damage is
detected among structurally diverse lesions and substrates by UvrA2B2? How UvrA
is dislodged from the repair complex and how does UvrB recruit UvrC? What is the
stoichiometry of UvrB and UvrC in the complex? What is the functional relevance of
UvrC homodimers and homotetramers? There exist significant differences in the
NER pathway between E. coli and other bacteria, but very less is known about NER,
especially in bacterial pathogens such as M. tuberculosis. Furthermore, many species
like M. smegmatis, Streptomyces species, D. radiodurans, and Pseudomonas putida
possess a second copy of UvrA, but its actual role in vivo is unclear (Timmins et al.
2009; Tark et al. 2008).
The deletion of Yersinia pseudotuberculosis and Borrelia burgdorferi uvrA genes
or disabling UvrA activity leads to their decreased virulence (Darwin and Nathan
2005; Garbom et al. 2004; Graham and Clark-Curtiss 1999). Additionally, the uvrA
gene transcript levels are upregulated in M. tuberculosis grown in human

macrophages (Graham and Clark-Curtiss 1999). Trans-complementation of
M. tuberculosis uvrB demonstrated a crucial role in mounting resistance against
nitrosative, oxidative, and UV exposure-induced DNA damage (Darwin and Nathan
2005; Graham and Clark-Curtiss 1999). M. tuberculosis uvrB has been shown to be
important for attenuation of virulence in iNOS wild-type and iNOS knockout mice
(Darwin and Nathan 2005). Notably, clinical isolates of XDR strains of
M. tuberculosis harbour a unique uvrBA582V mutation, an important (catalytic) active
site residue (Eldholm et al. 2014). An inhibitor, [2-(5-amino-1,3,4-thiadiazol-2-
ylbenzo[f]chromen-3-one) (ATBC)], against UvrABC complex works at a micro-
molar concentration, but the molecular basis of its activity and the specific target
within the complex has not been deciphered (Mazloum et al. 2011). Thus, efforts are
underway to identify M. tuberculosis key determinants of infection and vulnerable
targets from the NER system whose structures and biochemical functions could be
exploited for the development of new antitubercular agents (Ferraris et al. 2018).
Genetic analysis revealed that M. tuberculosis UvrA is important for survival
against DNA-damage, including nitrosative and oxidative DNA damage,
implicating this gene in the persistence of the bacillus in the host. Comparative
analysis of the crystal structures of M. tuberculosis UvrA in its ligand free form with
B. stearothermophilus UvrA•UvrB complex, B. stearothermophilus UvrA•ADP,
and Thermotoga maritima UvrA•DNA showed the conformational plasticity
displayed by UvrB binding domain of UvrA (Rossi et al. 2011). Altogether, com-
bined genetic, biochemical, bioinformatics, and structural investigations of
M. tuberculosis UvrA have provided significant insights into the mechanistic
details of UvrA from M. tuberculosis. In-depth biochemical characterization
revealed that the M. tuberculosis UvrB subunit behaves as a DNA-stimulated
ATPase also possessing an ATP-dependent DNA helicase activity (Fig. 16.5)
(Thakur et al. 2016). Recent structural and biochemical studies have demonstrated
direct interaction between M. tuberculosis UvrA and UvrB proteins in the absence of
any ligands and further suggested that this protein–protein interaction may be
exploited as a target for blocking NER in M. tuberculosis (Ferraris et al. 2018;
Lahiri et al. 2018; Singh 2017). Although biochemical and structural studies are
limited in the case of M. tuberculosis UvrC, it is identified as an essential gene for
survival in mouse model of infection in transposon mutagenesis screen, and
mutations in this gene result in increased sensitivity against UV and hydrogen
peroxide (Sassetti et al. 2003; Prammananan et al. 2012).
M. tuberculosis possesses two UvrD homologues: UvrD1 and UvrD2 (Cole
1998). M. tuberculosis UvrD1 is a DNA helicase with 30 –50 polarity and unwinds
nicked DNA substrates resembling NER intermediates but displays maximum DNA
unwinding activity on replication forks (Curti et al. 2007; Singh et al. 2010). The
strains in which uvrD1 was inactivated either alone or in conjunction with uvrA
showed increased susceptibility to UV irradiation, mitomycin C, RNI, and ROI.
Moreover, uvrD1 and uvrA uvrD1 mutants showed attenuation following infection
of mice either by aerosol or intravenous route (Houghton et al. 2012).
M. tuberculosis UvrD1 and UvrA also suppress DNA strand exchange catalysed
Fig. 16.5 Model depicting differences in the biochemical properties of MtUvrB and EcUvrB
subunit. Each line (red and blue colour) represents a DNA strand with polarity as indicated.
Experimental evidences support the notion that MtUvrB can unwind DNA replication intermediates
like splayed duplex or replication fork DNA structures in an ATP-dependent manner. In contrast,
EcUvrB neither hydrolyses ATP nor unwinds these structures of its own. EcUvrB needs EcUvrA to
activate its ATPase as well as helicase activity. (Figure adapted with permission from Thakur et al.
2016) [Copyright 2016 American Chemical Society]
by M. tuberculosis, M. smegmatis, and E. coli RecA suggesting molecular cross talks

with homologous pathway involved in chromosome stability (Singh et al. 2010).
Inactivation of uvrB and uvrD1 genes increased marker integration frequencies,
further suggesting a role for these proteins in other DNA transaction processes
beside NER (Guthlein et al. 2009). Additionally, mycobacterial Ku interacts with
UvrD1 in a genome-wide yeast two-hybrid screen and stimulates UvrD1 to catalyse
its ATP-dependent unwinding of 30 -tailed DNA. This interaction was further
supported by the formation of a stable ternary complex of UvrD1, Ku, and DNA
in the absence of ATP (Sinha et al. 2007). Altogether, these findings revealed
structural and functional cross talk between NHEJ and NER. UvrD2 is the product
of an essential gene which is comprised of N-terminal ATPase domain (Pfam00580)
typical of superfamily I helicases and C-terminal HRDC domain (Pfam00570)
typical of superfamily II helicases. These two domains are connected by a CxxC-
(14)-CxxC tetracysteine module, which is present in helicases from Actinomycetales
(Sinha et al. 2007). The C-terminal HRDC domain is not required for in vitro DNA
helicase and ATPase activity, whereas tetracysteine module (but not the
tetracysteines) is required for DNA unwinding. Surprisingly, the helicase activity of

a truncated UvrD2 lacking the tetracysteine and HRDC domain can be restored by
the addition of Ku protein (Sinha et al. 2007). These findings suggest that the
Ku-dependent unwinding activity together with unusual domain structure of
UvrD2 may have a biological function, distinct from that of M. smegmatis UvrD1.
In M. tuberculosis, complementation by N-terminal domain of UvrD2 was able to
compensate for the loss of uvrD2 deletion suggesting that the C-terminal domains
are not essential. Moreover, an ATPase dead form of UvrD2 protein was unable to
translocate along DNA and cannot compensate for lack of the wild-type protein.
These studies indicate that the role of UvrD2 is not to unwind DNA, but rather in
DNA translocation and protein displacement (Williams et al. 2011). Since myco-
bacterial genome is rich in GC content, it is predicted that many genes involved in
persistence and pathogenesis have G-quadruplex forming motifs in their promoter
region, which might regulate their expression (Perrone et al. 2017). More recently,
MtUvrD1 and MtUvrD2 were subjected to extensive in vitro biochemical studies,
which revealed that these proteins have the potential to resolve G-quadruplex
structures, implicating their important roles in maintenance of genome integrity
and gene expression via G-quadruplex DNA resolution (Saha et al. 2019). The
polA mutants of M. smegmatis displayed hypersensitivity to DNA damage induced
by UV irradiation and hydrogen peroxide. Using transposon site hybridization
(TraSH), it was found that polA gene is required for the optimal growth of
M. tuberculosis (Gordhan et al. 1996; Sassetti et al. 2003). Mycobacteria possess
ligA (encodes a NAD(+)-dependent enzyme), which is essential for its viability
(Korycka-Machala et al. 2007; Srivastava et al. 2005). In addition, M. tuberculosis
contains three additional ATP-dependent ligases, LigB, LigC, and LigD which are
shown to be nonessential for cell growth under in vitro conditions (Gong et al. 2004).
Interestingly LigD interacts with Prim-PolC in the presence of single nucleotide gap
containing dsDNA substrate. Deletion of ligD gene was found to be sensitive to
oxidative damage, suggesting the role of LigD in the excision repair other
than NHEJ.
16.9 Concluding Remarks
M. tuberculosis, an exquisitely adapted human pathogen, encounters hostile

environments during transmission, infection, and clinical latency. M. tuberculosis
encodes a complex array of enzymes of several lipid pathways, DNA metabolic
pathways, cell surface proteins, and regulators, which are essential for genome
stability and cell survival in infected host cells. The attenuation of virulence of
M. tuberculosis uvrB , uvrA, and uvrD1 mutant strains in mice suggests a possible
role for NER in pathogenesis. Although the genes encoding proteins involved in the
NER pathway are conserved between E. coli and M. tuberculosis, the observation
that NER components in tubercle bacillus exhibit significantly different mechanistic
properties from the E. coli UvrABC proteins is worthy of further investigation.
Additionally, it would be interesting to investigate the role of NER in the generation
of MDR and XDR strains of M. tuberculosis and phenotypic heterogeneity among

these strains. These studies might help to guide the development of more efficacious
treatments targeting the NER components of M. tuberculosis. In conclusion, we
envision that further research on the role(s) of NER components in tubercle bacillus
may provide insights into the processes critical for genome maintenance and new
opportunities for therapeutic intervention.
Acknowledgements K.M. acknowledges funding from J. C. Bose National Fellowship (DST,

New Delhi), Bhatnagar Fellowship (CSIR, New Delhi) and a grant (BT/CoE/34/SP15232/2015)
under the Center of Excellence from the Department of Biotechnology, New Delhi. M.T. was
supported by a Senior Research Fellowship [09/079(2548)/2012-EMR-I] from the Council of
Scientific and Industrial Research, New Delhi. We apologize to our colleagues whose work could
not be cited due to space constraints.
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Mesenchymal Stem Cells: A Hidden Arsenal
for Mtb Persistence, Resuscitation, 17
and Reactivation
Jaishree Garhyan, Bikul Das, and Rakesh Bhatnagar
Abstract
Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is a
global public health threat and remains the leading cause of death among infec-
tious diseases. The pathogen has developed strategies to survive inside and
outside diverse host cells under wide range of unfavorable conditions, leading
to persistence, and also undergo reactivation in the event of favorable conditions.
Several sources of endogenous reactivation are proposed, including specific host
cells that could harbor viable and dormant Mtb in the protective intracellular
compartments. However, it is not clear how these host cells could survive the
hypoxic/oxidative stress microenvironment prevalent in the post-drug treated
granulomas. Recently Das et al. identified human CD271+ bone marrow mesen-
chymal stem cells (CD271+ BM-MSCs) as niche host cells that can maintain
viable, non-replicating dormant Mtb. Most importantly, from patients who had
successfully completed anti-tubercular therapy, viable Mtb could be isolated from
J. Garhyan
Department of Microbiology, University of Massachusetts, Amherst, MA, USA
B. Das
Department of Stem Cell and Infectious Diseases, Kavikrishna Laboratory, Guwahati Biotech Park,
Indian Institute of Technology, Guwahati, India
Department of Stem Cells and Infectious Diseases, Thoreau Laboratory for Global Health,
M2D2, University of Massachusetts, Lowell, MA, USA
R. Bhatnagar (*)
e-mail: rakeshbhatnagar@jnu.ac.in

https://doi.org/10.1007/978-981-32-9413-4_17
302 J. Garhyan et al.
CD271+ BM-MSCs, and not in other types of cells including macrophages. The
follow-up study showed that Mtb could escape anti-tubercular drug therapy by
hiding intracellular to CD271+ BM-MSCs serving as source for endogenous
reactivation of TB. With these studies at the forefront, a novel model of granu-
loma reactivation could be proposed where CD271+ BM-MSCs harboring viable
Mtb migrate to inflammatory granulomas for disease reactivation. Moreover,
CD271+BM-MSCs also are able to resuscitate the viable but non-culturable
(VBNC) Mtb inside the granuloma to reactivate the disease. Studies show that
pulmonary tuberculosis (PTB) patients with culture negative sputum contain an
occult population of VBNC Mtb in their sputum. CD271+ host-Mtb interaction
studies suggest that these VBNC may be resuscitate back to culturable state by
CD271+MSCs. Study of mechanism of Mtb survival and resuscitation inside
MSCs is challenging and ongoing. Hypoxia may play an important role in the
survival and persistence of Mtb inside the MSCs in TB patients post therapy as
well in mice drug-induced dormancy model indicated by upregulation of
HIF-1alpha. Latent TB has been challenging and difficult to study because of
the dynamic capability of Mtb to adapt easily for over 200 years in vivo and
in vitro. Despite all the ongoing efforts, so far, targeting dormant TB seems
difficult. Although there are drugs which target cell wall synthesis, transcription,
translation, and ATPase activity in actively dividing stage, their efficacy against
dormant bacilli is unclear. Isoniazid treatment for 9 months is recommended for
latent or dormant TB; however, non-adherence to such long regimen is a problem;
moreover in light of current studies the efficacy of isoniazid against dormant
bacilli is questionable. Studies on Mtb residing inside MSCs may shed light into
new therapeutics to target non-replicating bacteria as well as VBNC Mtb and
prevent endogenous reactivation of the disease.
Keywords
Mesenchymal stem cells · Mtb persistence · Dynamic granuloma model ·
Resuscitation
17.1 Introduction
Mycobacterium tuberculosis (Mtb) causes the deadly disease tuberculosis (TB) in

millions of humans, killing nearly 2 million yearly, despite a vigorous host immune
response and months of effective drug treatment, which transform active granulomas
into fibrotic and hypoxic sterile granulomas. Mtb has the spectacular ability of long-
term persistence and reactivation despite vigorous host immunity and prolonged
anti-tubercular therapy (Bloom and McKinney 1999). Studies indicate that after
successful drug treatment, Mtb persists in a non-replicating dormant state in the
17 Mesenchymal Stem Cells: A Hidden Arsenal for Mtb Persistence. . . 303
sterile granulomas and/or extra-granuloma sites. Epidemiological studies indicate

that these endogenous dormant Mtb may cause disease recurrence. Although TB
recurrence may also occur due to exogenous re-infection with a new Mtb strain, the
reactivation of endogenous dormant Mtb is a significant source of recurrent
TB. Most clinical cases are not due to new infections but through reactivation of
dormant Mtb residing in LTBI hosts (Gengenbacher and Kaufmann 2012). Hence,
understanding the endogenous reactivation is imperative to develop novel manage-
ment strategies against recurrent TB. However, the source of endogenous Mtb
reactivation is not clearly known. Several sources of endogenous reactivation are
proposed, including specific host cells that could harbor viable and dormant Mtb in
the protective intracellular compartments. However, it is not clear how these host
cells could survive the hypoxic/oxidative stress microenvironment prevalent in the
post-drug-treated granulomas. Recently Das et al. identified human CD271+ bone
marrow mesenchymal stem cells as niche cells that can maintain viable,
non-replicating dormant Mtb (Das et al. 2013). Most importantly, in patients who
had successfully completed anti-tubercular therapy, viable Mtb could be isolated
from CD271+ BM-MSCs, and not in other types of cells including macrophages,
which was presumed to be the primary host cells for dormant Mtb. Moreover,
CD271+ BM-MSCs protects viable Mtb from anti-TB drug therapy. Mtb escapes
anti-tubercular drug therapy by hiding intracellular to CD271+ BM-MSCs serving as
a depot for endogenous reactivation. Therefore, a novel model of granuloma reacti-
vation could be proposed where CD271+ BM-MSCs harboring viable Mtb migrate
to inflammatory granulomas for disease reactivation (Fig. 17.1). Moreover, ongoing
studies show that CD271+BM-MSCs also are able to resuscitate the viable but
non-culturable Mtb inside the granuloma to reactivate the disease.
Fig. 17.1 Hypothetical model of CD271+ BM-MSC role in tubercular dormancy and reactivation
Schematic representation of a novel model of granuloma reactivation where CD271+ BM-MSCs
harboring viable Mtb migrate to inflammatory granulomas for disease reactivation. We propose that
CD271+ BM-MSCs travel from bone marrow through the circulatory system to the lungs as they
are CD271+ BM-MSCs like most stem cells are programmed to migrate to the inflamed organs
17.2 Latency, Reactivation, and Post-treatment Relapse

of Tuberculosis
Clinical Relevance of Stem Cell Niche Strikingly, about one-third of the world’s
population has latent TB, where people are infected by TB bacteria but not (yet) ill
with active disease and cannot transmit the disease. Approximately 5–10% of the
latently infected patients are estimated to develop TB at some point in their lifetime,
while 10–15% of patients who have undergone complete anti-TB drug treatment
show relapse. However, it is puzzling that relapse occurs in patients where lung
granulomas are found sterile (Sia and Wieland 2011; Lin et al. 2014). Some
autopsies detect viable bacteria, but these subjects may have had “active” disease
(Altaf Bachh et al. 2010; Lin et al. 2014; Shleeva et al. 2002). Additionally,
autopsies in the Cornell murine model tend to support the human autopsy findings
and the speculation that persistent bacteria could somehow appear in relatively
sterile granuloma to reactivate the disease. Human studies found evidence of
M. tuberculosis inside cells like adipocytes, macrophages, epithelial cells, endothe-
lial cells, etc. (Mayito et al. 2019; Neyrolles et al. 2006; Antony et al. 1983; Mariotti
et al. 2013). But so far, none of these studies found the viable Mtb; only molecular
footprints in the form of DNA could be detected (Sia and Wieland 2011; Lin et al.
2014; Shi et al. 2005; Shleeva et al. 2002). However, these findings do indicate that
there are extra granuloma sites for Mtb, yet the question remained to be answered
conclusively: Where do they reside? Do they need niche cells or not?
Extra-Granuloma Sites of Dormant Mtb There are numerous studies on extra-

granuloma sites that serve as homing sites for Mtb. Studies describe wide arrays of
sites like the lung epithelial cells (outside granuloma), extrapulmonary sites like
adipocytes, endothelial cells, macrophages, etc. which could be probable extra
granulomatous location of Mtb in dormant stage (Mayito et al. 2019). One study
concluded that the bacilli can persist intracellularly in the lung tissue without causing
clinical lesions (Grosset 2003). M. tuberculosis DNA was found not only in
macrophages but also in other non-phagocytic cells like type II pneumocytes,
endothelial cells, and fibroblasts (Mariotti et al. 2013). Neyrolles et al. found
adipocytes in adipose tissue act as host for Mtb DNA (Neyrolles et al. 2006).
Similarly, other studies also found Mtb DNA in extra-granuloma sites (Shi et al.
2005; Reece and Kaufmann 2012; Shleeva et al. 2002). In light of all the above
autopsy and other reports, more questions may be raised: If granulomas are sterile,
where do the persistent Mtb reside? Where is their protective niche? How many of
them are required to activate the disease? Moreover, if robust why did not Mtb reside
in the primary granuloma? And, why does the reactivation take place in the apical
part of the lung?
Persistent Non-replicating Phenotype At present neither in vitro nor in vivo

models of dormancy provide adequate argument for correlation between numeric
quantity and survival of persistent non-replicating Mtb and reactivation. The well-
known murine Cornell model of dormancy tends to support that perhaps persistent
bacteria are rare and not plenty. Real-time PCR analysis detected only 1000 bacteria
per ml tissue, but eventually, this small number of bacteria seems enough for
reactivation of the disease in the animal (Mukamolova et al. 2010; Russell 2007;
de Wit et al. 1995). So, it may be assumed that only a few bacteria are required to
reactivate the disease.
In human, due to a more vigorous immune response to primary infection, the number
of persistent TB bacilli is probably smaller in number, and most of the time they may
not be culturable in the laboratory. So far, the attempt to culture persistent Mtb from
patients remains unsuccessful. There are reports of culturing Mtb from latent TB
individuals, but whether these subjects had the quiescent phase of latent TB or the
active phase of latent TB could not be ascertained. None of these studies confirm the
phenotype of latent TB.
In vitro models do not take into account the host elicited immune response
responsible for persistence. The immune system shapes the phenotype and might
maintain an equilibrium between host defense and the number of bacilli
metamorphosing into non-replicative type (persisting) with prospects of reactivation
at a later time point. However, most animal models do not confer dormancy, and in
most models a severe acute infection develops and the animal eventually succumbs
to death. Here we address the two known animal dormancy models which are
instrumental for reactivation studies:
Cornell Model of Dormancy The most clinically relevant in vivo model is the
Cornell model. In this model, anti-TB drug treatment leads to elimination of almost
all the bacteria in the infected animal indicated by zero CFU from lung or other
tissues. However, on immunosuppression, lung granuloma is observed, “sleeper
cells” get reactivated, and the disease reappears (Scanga et al. 1999). In clinical
settings, TB patients go through couple of diagnostic tests and are prescribed several
anti-TB drugs for nearly a year that kills most of the bacilli. However, similar to the
Cornell model, after a few months, or year, in about 10% cases, the disease
reappears, but the bacilli remain sensitive to drugs, indicating that the relapse was
not because of drug resistance but because of reactivation of the persistent bacilli that
was not cleared by chemotherapy. These relapse cases undergo another round of
anti-TB drug treatment; however many of them do not survive. The Cornell model
closely mimics the relapse clinical cases confirming dormant or persistent infection
reappearing from a non-pulmonary site to pulmonary site.
Forsyth Model of Dormancy In this model a mutant of M. tuberculosis strain

(auxotrophic for streptomycin), 18b strain, is cultured with regular addition of strepto-
mycin for 4 weeks. On streptomycin withdrawal for a week, Western blot panels show
synthesis of increased amount of alpha crystalline by the bacilli, indicating conversion
to a non-replicative form. In other words, the M. tuberculosis strain 18b gets into
dormant phase on streptomycin withdrawal. Therefore, this mouse model could
represent the putative dormant infection model, where bacteria is presumed to stay
in a non-replicating dormant state (Das et al. 2013).
The two above animal models support niche-to-niche interaction hypothesis.

There appears to be niche-to-niche interaction from bone marrow to lung granulo-
matous tissue for persistent bacilli to reactivate the disease. It appears that not all
persistent bacteria are culturable or viable. Thus, the granulomas in autopsies may
have bacteria detected under microscope, but not all of those are culturable.
Post-drug treatment, where granulomas are relatively sterile, Mtb may employ a
“sleeper cell” strategy, where a few viable bacteria hide among extrapulmonary
healthy tissues for years, instead of hiding in the granuloma to be eventually killed
by the immune cells. Residing within a host cell that sustains Mtb viability is crucial
for Mtb survival, dormancy, and reactivation Macrophages which are known to be
the primary host for Mtb may not be suitable for Mtb survival and persistence and
dissemination. Only 1 out of 10 bacteria retain viability in macrophages. Thus,
macrophages may not be an ideal host for the long term persistent of Mtb. What
would be the ideal host then, the adipocytes, endothelial cells, or other host cells?
17.3 Significance of the Non-granuloma Model of Tubercular

Dormancy
In animal models, Mtb acquires a dynamic equilibrium where some bacteria con-
tinue to proliferate and some acquire a persistent phenotype or a non-replicating
state. These non-replicating M. tuberculosis (NRM) are supposed to reside inside the
foamy macrophages and then periodically replicate to initiate a fresh cycle of
replication and infection of fresh macrophages to keep a granuloma alive. This
model of persistence is called dynamic granuloma model. Dynamic granuloma
model considers high persistent Mtb which are eliminated by anti-TB drug treat-
ment. Dynamic granuloma model is also shown in Guinea pigs which form persis-
tent granuloma lesion containing high number of persistent bacteria surviving in the
necrotic core of the granuloma (Lenaerts et al. 2007, 2015). Moreover, it is assumed
that a similar situation may also occur in human, where active secondary granuloma
may contain both the replicating and non-replicating bacteria. Clinical studies show
that subjects with latent TB benefit from long-term treatment, therefore supporting
the dynamic granuloma model of latency in human.
However, extrapolating model of dynamic granuloma to human can be debatable.
The first argument against it is that animals succumb to primary infection, whereas
TB patients rarely die of primary infection due to strong immune system. Second,
clinical studies support that chemotherapy could eliminate persistent TB, but that
does not confirm that a number of persistent Mtb are high in those patients.
Moreover, no diagnostic test confirms that those subjects who were benefitted
from chemotherapy belonged to pure latent state (or the quiescent state of the latent
TB), instead of the early stage of reactivation (or active state of latent TB). It is
expected that subjects with the active latency would benefit from drug treatment,
because it is expected to have replicating bacteria in the granuloma. On the other
hand, subject with quiescent state of latency may not get any benefit, because the
existing TB drugs are not known to target the persistent dormant bacteria. Overall,
the basic question remains unanswered: Are persistent TB plenty or rare? Do they
replicate during the quiescent latent stage or not?
Reactivation of Non-replicating Dormant Phenotype It is known that LTBI

(latent tuberculosis infection) patients show negative sputum culture and smear,
positive IGRA (Interferon-Gamma Release Assays) or TST (tuberculin skin test),
and no clinical symptoms whatsoever. Reactivation of LTBI can happen during the
patient lifetime and can turn into active TB indicated by clinical symptom along with
positive sputum, IGRA or TST, and chest x-ray. The fundamental mechanism
involved in reactivation of latent/dormant tuberculosis is not very clear. This could
be largely attributed to the difficulty in developing and manipulating animal models
of latent tuberculosis. Although few studies have provided some insights on the role
of T lymphocytes, gamma interferon (IFN-γ), tumor necrosis factor alpha
(TNF-alpha), interleukin-12, reactive nitrogen intermediate (RNI), etc. on reactiva-
tion (Islam et al. 2004), further research is impending.
17.4 Bone Marrow-Mesenchymal Stem cell Niche of Tubercle

Bacilli
The correlation between Mtb and stem cells was discovered by Das et al. (Das et al.
2013). A population of Mesenchymal stem cells, CD271+ MSCs, harbor tubercle
bacilli in bone marrow in animals as wells as human patients. CD271 is a type of low
affinity nerve growth factor receptor (LNGFR), which serves as a co-receptor for
neurotrophins, a family of nerve growth factors. LNGFR is also a member of the
TNF receptor superfamily. Interestingly, a subset of naïve MSCs express CD271 and
are localized in the hypoxic niche of human BM. Das et al. developed an in vitro
culture system to maintain the naïve CD271+ BM-MSCs (Pal and Das 2017), where
knockdown of CD271 was found to reduce glutathione level (unpublished data)
suggesting that the receptor may be involved in regulating the cellular redox level.
Whether this putative redox regulating activity of CD271 allows Mtb to hide
intracellularly to this type of MSC is not yet clear. Subsequently, Garhyan et al.
found out that non-replicating Mtb could be recovered post-antibiotic treatment
(Garhyan et al. 2015; Beamer et al. 2014). These results showed that long-lasting
quiescent nature of the CD271+ BM-MSC could provide long-term shelter to Mtb in
spite of anti-TB drugs treatment. MSCs have properties which provide long-term
protective niche for long-term survival of the Mtb bacilli. The following reasons
support the correlation between stem cells and Mtb: (a) Stem cells are present in
human lung granuloma and they are capable of self-renewal. (b) MSCs have drug
efflux pumps to efflux out the antibiotics in order to protect the bacilli. (c) The fact
that MSCs are quiescent and have low reactive oxygen species also allow them to be
a perfect niche for Mtb. It is worth noting that undifferentiated MSCs harbor Mtb,
while differentiated ones don’t harbor the bacilli.
Although CD271+ BM-MSC population provides a protective niche to dormant

Mtb (5), there are two fundamental questions which remain unanswered. First, what
is the direct role of CD271 in making MSCs suitable and long-term stable hosts for
Mtb? Second, what is the significance of CD271 in Mtb reactivation in the lung?
High expression of the drug efflux pump ABCG2 in CD271+ BM-MSCS indicates
that CD271+ marker is linked with offering antibiotic protection to the pathogen
(Das et al. 2013). Expression of CD271 followed by CD271-responsive genes is
responsible for genes associated with DNA repair (in p53-dependent manner) and
drug response in stem cells (Redmer et al. 2017; Li et al. 2015). In a separate study,
Fan et al. demonstrated that lethal oxidative DNA damage occurs in nongrowing
Mtb cells treated with different bactericidal antibiotics (Fan et al. 2018). With these
two studies, it is speculated that since CD271 is needed for a proper p53-dependent
response to DNA-damaging drugs, it may offer protection to intracellular nongrow-
ing Mtb from lethal oxidation-induced DNA damage caused by bactericidal
antibiotics and thus may offer enhance protection from antibiotics. To address the
second question of significance of CD271 in Mtb reactivation, we should keep in
mind that forced differentiation of Mtb-infected BM-MSCs leads to loss of Mtb
viability and loss of CD271+marker (Das et al. 2013) indicating that BM-MSCs’
undifferentiated state along with CD271+ marker may be the requirement for Mtb to
remain dormant. Thus, we can refute the possibility of differentiation of BM-MSCs
and reaching various sites to reactivate the disease. This further leads to the specula-
tion that BM-MSCs have to travel in their undifferentiated state with CD271+marker
(stem state) to the site to reactivate the disease (Fig. 17.1). Moreover, since CD271
also has a role in migration to various tissue (Li et al. 2015), it may provide an edge
to the CD271+ BM-MSCs to disseminate Mtb to various organs and reactivate in the
most favorable environment. Additionally, human MSCs are known to express
several Toll-like receptors, NOD-2 and Rig-I (Khan et al. 2017), which can be
significant in Mtb protection. Studies show that human mesenchymal stem cells
internalize Mtb through scavenger receptors MARCO (macrophage receptor with
collagenous structure) and SR-B (a.k.a. CD36). Unlike macrophages, mannose
receptors on MSCs are not responsible for Mtb uptake (Khan et al. 2017). MSCs
do not allow Mtb replication due to its intrinsic nitric oxide.
In our evolutionary history, mammalian ancestors might have had a strong BM-
to-lung route of regeneration: Mtb transmitted to its replicating niche from its
protective niche through its depot BM-MSCs, and thus, only a few Mtb bacteria (~
60 Mtb infected cells in mice) (Garhyan et al. 2015) is successful in establishing
infection in mice. Indeed, such atavistic reactivation of ancient primitive defense
mechanism is anticipated (Chen et al. 2000; Das 2000). Hence, further studies may
shed more light about the remnant of BM-lung regenerative mechanism, which of
course will greatly benefit the infectious disease and stem cell regenerative
research alike. It is important to note that recently hematopoietic stem cells have
also been found to be depot for dormant Mtb (Gengenbacher and Kaufmann 2012),
further supporting potential evolutionary aspect of the stem cell basis of TB latency
and reactivation.
Resuscitation of Non-replicating Dormant Phenotype by BM-MSCs We found

that PTB patients (from northeast region of India) treated with anti-TB drugs have
viable but non-culturable (VBNC) Mtb in sputum that show culturability only in the
presence of EPSN (resuscitation factor containing supernatant of mid log phase
H37Rv culture). Importantly, VBNC Mtb resuscitation can also be attained and
enhanced by culturing in CD271+BM-MSCs (unpublished work).
Pulmonary tuberculosis (PTB) worldwide is diagnosed by identifying the pres-

ence of acid-fast bacillus (AFB) under microscopy. Mtb culture from expectorated
sputum or tissue is the confirmatory gold standard test for active TB (Sia and
Wieland 2011). However, there is a huge pool of sputum smear-negative PTB
patients who show chest x-ray abnormal/positive while having a negative sputum
smear/culture. Sputum-negative tuberculosis poses a challenge in diagnosis, treat-
ment, and eradication of tuberculosis (Granville et al. 1953; Wayne and Sohaskey
2001; Hobby et al. 1954; Beck and Yegian 1952; Falk et al. 1954; Hurford and
Valentine 1957; Salkin and Wayne 1956; Dutt and Stead 1994; Mc 1959). Numer-
ous reports have shown the presence of non-culturable tubercle bacilli in human
sputum (Mukamolova et al. 1998; Garton et al. 2008; Gao and Fan 1986; Dhillon
et al. 2014) which are undetectable through culture and/or smear. CDC (Center for
Disease Control and Prevention) reports that only 50–80% of PTB cases are sputum
smear positive (Dooley et al. 1990). Other studies indicate that in India 28.7% of TB
cases are new smear-negative cases (Vengattaraman 2010; Altaf Bachh et al. 2010;
Ministry of Health and Family Welfare 2007). “With the sharp rise of PTB in
countries which are worst affected by the HIV epidemics, the number of patients
with suspected PTB who are sputum smear negative and chest x-ray/radiography
(CXR) positive has increased” (WHO, Geneva. TB Control as an Integral part of
primary health care. 1988, 11). The non-culturable Mtb in human tissue are
non-replicating, non-susceptible to chemotherapy with added ability to resuscitate
back to culturable cells, remaining potential source of reactivation of the disease
(Mc 1959; Russel et al. 1955).
Reactivation of dead and sterile granuloma is a major issue in pulmonary
tuberculosis management (Lin et al. 2014). The relevance of VBNC Mtb and the
mechanism of VBNC cell reactivation in the latent TB remain unknown to most
extent. Understanding the VBNC reactivation mechanism may provide new insight
about latent TB, and developing new therapy. Most of the latent TB granuloma are
sterile as indicated by autopsy studies in primates (Hobby et al. 1954; Lin et al. 2014;
Shi et al. 2005). In cases where Mtb is recovered from latent granulomas, they are
considered to be in VBNC state (Gengenbacher and Kaufmann 2012; Reece and
Kaufmann 2012). Numerous studies show that addition of spent media from expo-
nentially growing Mtb resuscitates the non-culturable bacteria culturable (Shleeva
et al. 2002), suggesting that the sterile granuloma contains viable but non-culturable
(VBNC) Mtb, also known as persisters. VBNC state is a general adaptive strategy of
many bacteria that evolved specific resuscitation-promoting factor genes, rpf
including Mtb (Gengenbacher and Kaufmann 2012) (Reece and Kaufmann 2012).
Strikingly, RPF-dependent VBNC Mtb were recovered from sputum of patients with
active TB and also from autopsies indicating the potential clinical significance of
VBNC Mtb (Mukamolova et al. 2010). Although extraneous addition of RPF may
resuscitate VBNC cells in vitro, the unavailability/inaccessibility of RPF in latent
granuloma could be argument against the hypothesis that the latent granuloma are
physically separated by fibrous wall or caseum and devoid of actively replicating
bacteria (Gengenbacher and Kaufmann 2012; Russell 2007). However, as Mtb is
heterogeneously persistent in different dynamic stages, the slowly-replicating ones
(Gong et al. 2015; Lenaerts et al. 2015; Cadena et al. 2017) may be able to produce
RPF for the replication of VBNC bacilli in hypoxic/necrotic granuloma. Reactiva-
tion occurs even in immunocompetent individual with LTBI where lungs are
paucibacillary Mtb state (Dutta and Karakousis 2014) through unknown mechanism
(Nunes-Alves et al. 2014; Stewart et al. 2003). To add to this complexity, VBNC
Mtb is highly immunogenic and, therefore, may pose additional challenge for the
survival of resuscitated VBNC Mtb in immunocompetent subjects. Hence, it could
be presumed that a yet unknown mechanism may resuscitate the VBNC Mtb in latent
granuloma by evading immune system.
VBNC can be studied in in vitro by Wayne model where Mtb cultured under
hypoxia (to mimic tubercle bacilli in vivo) progressively switches to two culturable
states of non-replicating persistence, NRPI and NRP II (Wayne and Hayes 1996;
Wayne and Sohaskey 2001). On longer incubation, NRP II progresses further to true
dormant bacteria requiring resuscitation to return back to culturable phenotype
(Wayne and Hayes 1996; Wayne 1977), closest to VBNC state in tissues. This
viable, non-replicating dormant bacteria can be converted to replicative form using
enriched media. For example, early stationary phase culture supernatant of Mtb has
resuscitation activity which could resuscitate dormant bacilli in old batch cultures.
Zhang et al. showed that dormant Mtb could be resuscitated in solid plates, using
culture supernatant of TB bacilli (Sun and Zhang 1999). VBNC in vitro model may
add on tools to solve the puzzle of reactivation.
17.5 Stem Cells and Tuberculosis Therapy
All past, present, and ongoing studies indicate that stem cells can either reactivate
TB or protect from the disease. One study shows that autologous infusion of stem
cells acts as an adjunct therapy for MDR and XdR Mtb patients (Gengenbacher and
Kaufmann 2012). When administered with second-line drugs, stem cell therapy
improves the clinical condition of the patients. Moreover, hematopoietic stem cells
(HSCs) have recently been shown to play an important role in protective immunity
against Mtb (Kaufmann et al. 2018). Divangahi et al. interestingly found that
Bacillus Calmette-Guerin (BCG) exposure to bone marrow HSCs can epigenetically
modify the macrophage progeny from the BCG-educated HSCs (Kaufmann et al.
2018). More importantly, these epigenetically modified macrophages are far better in
protection against Mtb compared to the unmodified ones. This study also suggests
that stem cells of BM specifically hematopoietic in origin can be used for vaccine
development. Overall, bone marrow-residing stem cells, both mesenchymal and
hematopoietic, provide promising novel strategy for treatment and/or prevention of
pulmonary tuberculosis.
Summary Mtb has co-evolved with human host and therefore has several strategies
to survive against vigorous immune reaction. Mtb-host coexistence for decades can
be either symbiotic where each is supporting the survival of the other or a perpetual
race against each other where one has to survive over the other. Bone marrow stem
cell niche is emerging as an important niche for Mtb dormancy and reactivation.
Recently Das et al. identified human CD271+ bone marrow mesenchymal stem cells
(CD271+ BM-MSCs) as niche host cells that can maintain viable, non-replicating
dormant Mtb. Moreover, CD271+BM-MSCs also are able to resuscitate the viable
but non-culturable (VBNC) Mtb inside the granuloma to reactivate the disease.
Study of mechanism of Mtb survival and resuscitation inside CD271+ BM-MSCs
is ongoing. In CD271+ BM-MSC, hypoxia-responsive genes (HIF-1alpha signaling
pathway) along with DNA damage-responsive genes (CD271 signaled
p53-dependent signaling pathway) may play an important role in the Mtb survival
and persistence. The self-renewing and metabolically quiescent plus low ROS state
of CD271+ stem cells may provide a favorable environment for the pathogen to
remain in the CD271+ BM-MSC niche. In addition to MSCs, other stem cell types in
BM niche, including HSCs, may also provide host support for Mtb dormancy and
reactivation.
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Biofilms: A Phenotypic Mechanism
of Bacteria Conferring Tolerance Against 18
Stress and Antibiotics
Anwar Alam, Ashutosh Kumar, Prajna Tripathi,

Nasreen Z. Ehtesham, and Seyed E. Hasnain
Abstract
Biofilm, as a heterogenous congregation of microbial cells enclosed within a
pellicle, has largely gained attention due to their historical importance in environ-
ment as sludges, flocs, slimes, etc. Biofilms in medical research have been an
active area of research in periodontics, in wounds, and in surgical implants. With
the availability of whole genome sequences, it is now evident that the
mechanisms that control biofilm formation have largely remained conserved
during the course of evolution, pointing to the fact that biofilm formation is an
Anwar Alam, Ashutosh Kumar and Prajna Tripathi have contributed equally to this chapter.
A. Alam
A. Kumar
Department of Microbiology, Tripura University (A Central University), Agartala, Tripura, India
P. Tripathi
N. Z. Ehtesham
S. E. Hasnain (*)

https://doi.org/10.1007/978-981-32-9413-4_18
316 A. Alam et al.
integral part in the lifecycle of any unicellular organism. The ability to easily inter-
switch between planktonic to a sessile life cycle is an important armor for these
unicellular organisms to overcome stress. The matrix not only acts a physical
barrier that protects the bacteria but also provides an ecological niche for close
interaction and communication among these unicellular entities. This coordinated
community-like behavior synchronizes metabolic upregulation or downregulation,
both in time and space, and allows these microorganisms to achieve physiological
proficiency in terms of ability to tolerate stress that might not be possible as a single
cell. Research on biofilms from the perspective to explore the mechanisms of drug
tolerance is now considered an apt model as compared to the use of planktonic
microorganisms. The increasing use of medical implants further necessitates the
need to accelerate research in anti-biofilm strategies for these medical devices. This
chapter presents an overview of the mechanisms of biofilm formation and the
various interventions for prevention of biofilms.
Keywords
Biofilm · Persister · Drug tolerance · Mycobacterium tuberculosis
Abbreviations
ABC ATP-binding cassette transporters

CaCl2 Calcium chloride
cAMP Cyclic adenosine monophosphate
FDA Food and Drug Administration
GMP Guanosine monophosphate
kHz Kilohertz
MgSO4 Magnesium sulfate
NaCl Sodium chloride
RNA Ribonucleic acid
18.1 Introduction
Microbial biofilm refers to assemblage of monospecies or poly-species microbial

cells that is irreversibly attached with surface or with each other and is embedded
within an extracellular matrix of polymeric substances. Cells within the microbial
biofilms exhibit an altered phenotype in terms of growth rate or gene transcription
and are therefore distinct as compared to the colonies of bacteria growing on agar
plate macromolecular polymer matrix.
Antony van Leeuwenhoek is credited with the first account of biofilm when he
reported (1683–1708) the presence of aggregated microbes in the scurf of his teeth
and tongue. Bacterial adhesion to surfaces is a common phenomenon that has routinely
been observed in the environment. The term film has been used in marine microbiology
18 Biofilms: A Phenotypic Mechanism of Bacteria Conferring Tolerance Against. . . 317
to distinguish sessile microorganisms from the planktonic forms. The “bottle effect”
observed for marine microorganisms pointed that bacterial growth and activity increased
significantly due to attachment of these organisms on the surface. Initial investigation
about the composition of biofilm using “ruthenium red” dye followed by fixation with
osmium tetroxide showed that biofilms consist of polysaccharides. The medical rele-
vance of biofilm emanated from the studies carried out on sputum samples of patients
suffering from lung infection due to cystic fibrosis (CF) caused by Pseudomonas
aeruginosa. The first image of medical biofilms, published in 1977, was obtained
from lung biopsies and showed aggregated bacteria surrounded by “glycocalyx.” The
term glycocalyx was soon replaced with “biofilms” to emphasize the in vivo sessile
growth of colonized microorganisms that were considered to cause antimicrobial resis-
tance as compared to planktonic microorganisms (Costerton et al. 1987). The focus of
research that was initially directed toward testing medicaments on planktonic forms of
microorganisms soon diverged as it was observed that organisms in the biofilms showed
altered physiology and gene expression. Nearly 80% of the hospital-borne infections is
attributed to biofilm-forming microorganism and hence studies on biofilm-forming
microorganism have taken the front seat of all major research on drugs (Kumar et al.
2017). The major thrust in biofilm research has been due to the advent of instrumentation
such as scanning electron microscopy (SEM), confocal laser scanning microscopy,
Robbins device biofilm sampler, etc.; novel techniques such as that have complemented
standard microbiological culture techniques for biofilm characterization.
Bacteria ubiquitously form biofilm and the mechanism of adhesion is highly
conserved across different species of bacteria during the course of evolution. Biofilm
formation is an inherent and distinct phenomena among bacteria surviving in the
environment, where adhesion and protection is of higher importance and hence a sessile
life cycle is favored. The evolution of nonpathogenic environmental bacteria to patho-
genic strains has led to shedding of the biofilm machinery and enhancement of motility
machinery required for pathogenicity-invasion of host cells and escape from immune
cells. This is evident from the observation that subculturing of planktonic laboratory
strains of bacterium leads to shunting off of the genetic machinery involved in expres-
sion of cellular components required for adhesion, thereby rendering these cells inca-
pable to form biofilms. In the presence of selective pressure such as surfactants and
antibiotics, genetic machinery essential for formation of protective surface glycocalyx is
triggered. Cells with surface-bound glycocalyx continue to survive while planktonic
cells lacking protective surface structures fail to survive. The interaction of bacteria with
the environment, within and outside the community, and the outcome of the response
toward drugs are predominantly influenced by the phenotype as well. In drug discovery
strategies, biofilm model could provide more relevant data that may address core
questions as compared to the use of planktonic laboratory strains.
18.2 Biofilm Formation by Bacteria
Bacterial species are known to inhabit a variety of ecosystems and persist even under
extremely adverse conditions. The survival mechanisms of some pathogenic
microbes can be clearly credited to their ability to form biofilms. In addition to
318 A. Alam et al.
acting as a physical barrier against shear forces and antimicrobial agents generated
by the host system, biofilms also provide a protective niche for the survival of
bacteria (Jefferson 2004). Bacteria use their ability to form biofilms as a defense
strategy against host-mediated assaults like perturbations in pH, generation of
reactive oxygen intermediates, nutrient deprivation, attack by phagocytic cells, etc.
Biofilms are considered as the product of evolution against adverse conditions. The
fact that certain stress regulators and chaperones significantly contribute to the
phenomenon of biofilm formation indicates that a major motivation behind biofilm
formation is defense. Stress response mediators such as σB, RpoS, GroEL, DnaK,
and DnaJ have been implicated in microbial biofilm formation. In addition to stress,
there are other factors as well that trigger the formation of such a sessile microbial
community. Pathogenic and commensal bacteria also use biofilm development as a
means to colonize and persist in environments that seem propitious within the host
and thus flourish well with time. Bacterial species such as Staphylococcus, Strepto-
coccus, Pseudomonas, Vibrio cholerae, etc., display elaborate biofilm development
under conditions that offer readily utilizable carbon sources in abundance suggesting
that this may also act as a trigger factor. Biofilms formed at high shear locations,
such as blood or saliva stream, are highly viscous and display remarkable tensile
strength, thus conferring tremendous survival advantage to the constituent microbial
cells. Moreover, microbes also reap other benefits from this interactive community
such as division of metabolic labor. Close proximity of microorganisms within the
biofilm also fosters horizontal exchange of genetic information as well as imparts
reproductive advantage to these organisms and may eventually act as a driving force
for biofilm formation. Taken together, the community approach of growth endows
microorganisms with many benefits and there may be different forces that motivate
them to enter into this state.
18.3 Process of Biofilm Formation
The formation of this close-knit community of bacteria called biofilm is a complex

process involving multiple steps (Fig. 18.1). The series of events that occur during
this process, which eventually leads to adaptation under diverse environmental and
nutritional conditions, include (i) initial adherence to surface (ii) microcolony forma-
tion (aggregation and encapsulation), (iii) formation of a three-dimensional matrix
and maturation, and (iv) detachment and dispersal. Initiation of each of the above
stages may be contingent upon regulation of the expression of different sets of genes.
Biofilm development is initiated by a reversible attachment of motile bacterial
cells to a biotic or abiotic surface. This initial adherence is mediated by physical
forces like hydrophobic interactions, van der Waals forces, etc., and thus is a
noncommittal stage where microbial cells can detach themselves from the surface.
Various environmental and genetic factors contribute to the process of adhesion.
Bacteria cells may choose to come off if the surface doesn’t provide requisite
anchorage or nutrients to them or if other variables such as bacterial orientation,
local temperature, pressure, etc., do not seem favorable. However, once the cell
Fig. 18.1 Process of biofilm formation. Planktonic forms of bacteria reversibly attach to the
substratum and release quorum sensing molecules that allow them to irreversibly aggregate. The
sessile bacteria endogenously secrete matrix around the cells, which provide a niche for growth and
cell division. In presence of stress, such as drugs, few bacteria cells undergo dormancy and become
non-replicating persister cells. Within the mature biofilm the cells communicate and show
community-like behavior. As soon as stress is withdrawn, the dormant bacteria become active
and are released as planktonic forms to start the next cycle of biofilm formation
surface structures like pili, fimbriae, flagella, etc., come into play, the bacterial cells
become immobilized and proceed to a state of irreversible attachment. Evidence
shows that microbial adhesion extensively depends on the surface properties of
substratum. Additionally, it is reported that the bacterial appendages promote elec-
trostatic interactions and chemical reactions in order to consolidate the bond between
surface and bacterial cells, e.g., surface pili type IV, have been implicated in
adherence and movement of P. aeruginosa colonies through viscous cell surfaces
(Klausen et al. 2003). Attachment of bacteria to biotic surfaces is largely governed
by the interaction of host surface proteins and extracellular carbohydrate moieties
with that of the invading bacterial cell. E. coli is reported to adhere to host cells using
its pili type I adhesions like FimH that bind to mannosylated moieties on the surface
of host (Wright et al. 2007). Similarly, Enterococcus species have been shown to
adhere to host cells and aggregate further with the help of adhesion molecules like
Sag (Mohamed and Huang 2007).
Irreversible adhesion is followed by aggregation of bacterial cells leading to the
formation of a microcolony. This results in multiplication of bacteria and coloniza-
tion of the site. The formation of such a community demands for the development of
methods to communicate and exchange information between the constituent micro-
bial cells. Crosstalk between bacterial cells induces the production of
exopolysaccharides, signaling molecules, and peptides that enhance structural integ-
rity of the biofilm and coordinate bacterial growth via quorum sensing. Quorum
sensing can be described as a mechanism where a variety of autoinducers stimulate
genetic expression of regulators of cell density.
320 A. Alam et al.
The stimulation of cell signaling molecules and selective regulation of certain

genes responsible for extracellular polysaccharide (EPS) secretion triggers the for-
mation of a three-dimensional extracellular matrix. EPSs have been found to play a
pivotal role in determining the phenotype of a bacterial biofilm. Many bacteria use
cyclic-di-GMP as the second messenger for production of various EPS components
of the matrix. Cellulose has been shown to be an important component of the
extracellular matrix formed by E. coli and Salmonella typhimurium. The complex
three-dimensional architecture of a biofilm has channels that aid in the uptake of
nutrients and release of waste material. The Staphylococcus species produce poly-
saccharide intercellular adhesin (PIA) and poly-N-acetyl glucosamine (PNAG)
polymers as the major components of their biofilm matrices, whereas, in the case
of P. aeruginosa, polysaccharides such as Psl, Pel, and alginate are the main
constituents of extracellular matrix (Orgad et al. 2011). Thus, great diversity exists
in the phenotype and nature of different bacterial biofilms. Similarly, dramatic
differences in the matrix architecture may result from slightest of environmental
perturbations. Other constituents of the EPSs may include proteins, amyloids, DNA,
lipids, etc.
The concluding stage of biofilm development is characterized by detachment of
cells leading to dispersal of bacteria. Bacteria switch from sessile to motile lifestyle
either by passive sloughing due to fluid shear or by specific dispersal due to nutrient
limitations, environmental cues, and other signals. These signals stimulate the
expression of EPS-degrading enzymes and motility structures while downregulating
the genes mediating bacterial adherence (McDougald et al. 2012). Detached cells
may become quickly adapted to planktonic life or may disseminate further to
colonize newer sites.
18.4 Factors Involved in Regulation of Biofilm Formation
The initiation of the complex process of biofilm development relies largely on the
dynamics of bacterial attachment to a substratum. Bacterial cell surface properties
such as hydrophobicity, presence of motility appendages, and surface molecules
play an important role in initial attachment to a surface. Certain features of a surface,
such as roughness, may favor biofilm formation. Rough surfaces present higher
surface area and arrest shear forces. The rate and extent of bacterial adherence may
also be affected by the physicochemical properties of a surface. Interaction with
hydrophobic nonpolar surfaces results in stronger attachment as they appear to
enable bacteria to defy repulsive forces.
Fluctuations in the nutrient pool or ionic concentrations in the biotic or abiotic
habitat of bacteria may alter the properties of biofilm. In some strains of Lactobacil-
lus, nutrient limitation (especially low carbon to nitrogen ratio) may trigger biofilm
formation. Contrary to this, in P. aeruginosa nutrient deprivation induces detach-
ment of the biofilm. Similarly, availability of peptides and amino acids in host tissues
and body fluids has been reported to be crucial for several bacteria to form biofilm.
Biofilm formation in Listeria monocytogenes proceeds only in the presence of

carbohydrates like mannose and trehalose and appropriate levels of phosphate in
the local environment. The presence of salt such as NaCl, MgSO4, and CaCl2 in
appropriate concentrations has also been reported to be crucial for biofilm formation
(Marsden et al. 2017).
Environmental factors also play a significant role throughout the process. The
concentration of second messengers such as cyclic-di-GMP and cAMP, crucial for
surface attachment of bacteria, is controlled by the levels of carbon and oxygen in the
local environment (McDonough and Rodriguez 2012). Perturbations in the environ-
mental conditions result in dynamic changes in biofilm structure; e.g., under oxygen-
rich conditions P. aeruginosa displays a mushroom-shaped morphology with water
channels between macro-colonies consisting of rodlike cells, whereas, under anoxic
conditions, it has a three-dimensional mesh-like morphology with channels between
macro-colonies of elongated filamentous cells. These morphological changes possi-
bly alter the diffusion of nutrients and aid the bacteria to adapt metabolically.
Insufficient oxygen may abrogate the process of biofilm formation in E. coli.
Temperature is reported to be an important factor for the regulation of EPS produc-
tion in Clostridium perfringens and hence it dramatically affects the biomass,
density, and thickness of the biofilm. Similarly, L. monocytogenes loses its ability
to form biofilms at higher temperature. In addition to temperature, variations in pH
have also been shown to influence biofilm formation in the clinical isolates of
Burkholderia pseudomallei. While alkaline environment is required by Vibrio
cholerae cells to multiply and form biofilm, in the case of Lactobacillus rhamnosus,
biofilm formation is modulated by the presence of bile and mucins, low pH, and high
osmolarity (Lebeer et al. 2007). Overall, such changes in the morphology of bacterial
biofilms may be seen as a fitting response to the environmental cues and a practice
for better adaptation to the surrounding environment.
The intrinsic biofilm formation ability is also contingent upon genetic factors.
Studies on the regulatory mechanism driving bacterial biofilm formation have
identified several genes such as the flagella regulator sinR in Bacillus subtilis and
transcription activator prfA in L. monocytogenes as the major orchestrators of the
process (Newman et al. 2013). Becker et al. (2001) reported that Staphylococcus
aureus residing in biofilms showed enhanced expression of enzymes such as phos-
phoglycerate mutase and alcohol dehydrogenase that are involved in glycolysis and
fermentation. In addition, the transcript levels of two other genes sarA and sigB were
found to be distinctly upregulated in S. aureus biofilms. Bacteria deficient in sigB
were found to have lost the ability to form biofilm. Gene expression studies in E. coli
biofilms revealed enhanced expression of ompC and slp, the genes encoding outer
membrane protein and lipoprotein, respectively, which have recently been
associated with the initial steps of E. coli biofilm formation. The microarray analysis
of biofilms also exhibited differential expression of genes under hypoxic and
nutrition-deprived conditions. RpoS, the σS subunit of RNA polymerase, is consid-
ered to play a pivotal role in biofilm formation. Majority of the differentially
regulated genes identified till date are involved in bacterial motility, chemotaxis,
attachment, membrane bioenergetics, glycolysis, and phage-related functions.
322 A. Alam et al.
18.5 Interaction of Microbial Community Within Biofilms
Within the biofilm, bacterial cells can coexist with members of same species of
bacteria or with other species of microorganisms. The metabolic potential of each
species of bacteria govern the relative composition of bacteria within the biofilms.
The substrate concentration can influence the population of the bacteria; the fast-
growing population of bacteria can dominate over the slow-growing bacteria. The
interaction between cells may be competitive in terms of nutrient, space, metabolites,
etc.; however in certain conditions cells may act cooperatively to facilitate survival
of bacteria from same or other species. The distribution of bacterial population is
also dependent on oxygen tension; the core of the biofilm is dominated by cells that
are anaerobic while the outer layer of biofilms support aerobic bacteria. This spatial
distribution of cells within biofilms is evident in the case of biofilms inhabitated by
aerobic Burkholderia cepacia and facultative aerobic Klebsiella oxytoca. At an
initial microcolony formation stage when the biofilm matrix is minimum,
B. cepacia dominates the population due to its higher growth rate. As the biofilm
matrix accumulates it hinders diffusion of oxygen and creates a gradient of oxygen
tension. Under low oxygen tension, K. oxytoca gains advantage and grows at a faster
rate as compared to B. cepacia. Bacteria produce toxins and metabolites that confer
them with competitive edge over inter /intraspecies of bacteria and allow them to
gain foothold within the matrix. P. aeruginosa species can produce growth inhibi-
tory toxins that allow it to invade the biofilms caused by B. cepacia and eventually
displace the competitor cells. When both species of bacteria produce inhibitory
toxins, then the net effect on competitive survival of cells depends on timing of
release of toxins and the relative toxicity response of bacteria from each species. In a
mixed biofilm, toxins produced by Pseudoalteromonas tunicata suppress the growth
of sensitive species like Cytophaga fucicola but not of insensitive species such as
Roseobacter gallaeciensis. The protective efficacy of biofilms is enhanced due to the
synergistic presence of multiple species of bacteria within biofilms (Tolker-Nielsen
2015). A study showed that marine bacteria, when co-cultured with other species of
bacteria, achieved nearly twofold higher levels of survival within biofilms as com-
pared to monospecies (Burmølle et al. 2006). Staphylococcus aureus and Candida
albicans are often found to coexist in biofilms and exhibit higher virulence and
tolerance to antimicrobial drugs such as vancomycin. C. albicans is usually absent
on teeth of health individuals and does not cause symptomatic disease. However,
when C. albicans partners with dental bacteria such as Streptococcus gordonii or
S. oralis, it enhances bacterial aggregation, colonization, and biofilm formation.
These cross-kingdom interactions between fungi and bacteria can be either coopera-
tive or competitive and mediate synergistic or antagonistic associations that are
further modulated by host immune responses and local environmental factors.
18.6 Antibiotic Resistance of Bacterial Biofilms
Majority of the pathogens and environmental organisms are found to be present in

biofilm and as single entity in nature. Majority of the microbial and chronic
infections are associated with biofilms that increase chances of nosocomial
infections. Bacterial biofilm resists phagocytosis and immune response on
pathogens. It is known that the aquatic biofilms are reservoirs of resistant bacteria
that present very close to each other increase chances of genetic exchanges such as
antibiotic resistance genes (Balcázar et al. 2015). There are bacterial biofilms present
on the root and other parts of plants that may protect from plant pathogens (Bogino
et al. 2013). It has been reported that during maturation of biofilms there are
increases in the expression of genes related to energy metabolism and transporter
genes whereas decrease in the expression of genes related to cell structure, central
intermediary metabolism, energy metabolism, nucleotide biosynthesis, and metabo-
lism that indicates that biofilm cells are metabolically active but retard its cell
division. There are abnormal conditions present inside biofilms where depletion of
oxygen concentration, slow growth, increased doubling times due to nutrient limita-
tion, gradients in pH, change in the metabolic activity of microorganisms, presence
of compounds that may inactivate antibacterial compounds, and slow diffusion of
antibiotics make microorganisms resistant to antibiotics and drugs. The antibiotic
resistance in biofilms may be divided into innate biofilm factors such as oxidative
stress, microenvironments within biofilms, biofilm matrix as a diffusion barrier, etc.,
or induced resistance factors such as changes in the protein expression at transcrip-
tional level, stress protein expression, increased horizontal gene transmission
resulting in drug resistance, etc. Reduced antibiotic susceptibility due to biofilm
formation at the implanted devices causes persistent infections. It has been reported
that P. aeruginosa isolates from cystic fibrosis (CF) patients are sensitive to
antibiotics in planktonic form but difficult to eradicate due to the presence in biofilms
(Hengzhuang et al. 2012). In the case of tuberculosis, M. tuberculosis face unfavor-
able conditions inside hosts that are similar to biofilms that increase the chances of
drug tolerance by pathogen.
18.7 Tolerance to Antibiotics and Efflux Pumps
More than 80% of nosocomial infections are related to biofilms that may be present
at the medical devices. In the biofilms, bacteria encounter several types of stresses;
because of that there are upregulation of toxin-antitoxin systems, different stress
proteins, proteins involved in growth regulation, and downregulation of metabolic
proteins that causes tolerance to antimicrobial drugs. There are bacteria of different
physiological states present in the biofilm due to differential gene expression
resulting in differences in the susceptibility of antimicrobial compounds. There are
324 A. Alam et al.
different mechanisms that may be activated during drug tolerance such as modifica-
tion in drug target site, inactivation of drugs, upregulation of efflux pumps, etc. It is
known that expression of efflux pumps found in both bacteria and eukaryotic
organisms changes in response to different physiological and environmental
conditions (Sun et al. 2014), and these pumps may efflux antibiotics, heavy metals,
pollutants, chemicals involved in quorum sensing, compounds produced by plants,
bacterial metabolites, etc. (Blanco et al. 2016). In the biofilm, there is activation of
efflux pumps and stress response toxin-antitoxin systems that increase tolerance to
drugs, so these may be effective targets for anti-biofilm strategies. It has been
reported that there are different efflux pumps in P. aeruginosa that are involved in
resistance to different antibiotics such as ciprofloxacin, gentamicin, and tobramycin.
MacABCsm, an ABC-type tripartite efflux pump, is involved in drug resistance and
biofilm formation in Stenotrophomonas maltophilia. The multidrug resistance efflux
pumps of Salmonella enterica serovar Typhimurium are involved in biofilm forma-
tion and drug resistance. There is increase in the cases of drug resistance in the
tuberculosis infection. M. tuberculosis survives inside macrophages that induces
efflux pumps resulting in increases in drug tolerance and treatment duration (Adams
et al. 2011). Inhibition of these efflux pumps may be effective strategies to decrease
treatment duration.
18.8 Impact of Biofilm Formation and Phenotypic Behavior
The genes that regulate biofilm formation have largely remained conserved along the
course of evolution, pointing to the fact that biofilm formation is an essential
component in the life cycle of the unicellular organism. Biofilm formation offers
an ecological niche for the unicellular microorganisms to come in contact with each
other and offers the unicellular organisms with a great chance to adopt a lifestyle
where the functions of many cells cooperatively act to drive across a process that
might be impossible to carry as a single cell. The close proximity of unicellular
microorganisms within the matrix allows cells to transfer genes through conjugation.
It has been established that the conjugation rates in biofilms are as high as 1000-fold
as compared to planktonic forms (Stalder and Top 2016). Biofilms are thus consid-
ered “hot spots” for horizontal gene transfer and allow bacteria to evolve and adapt
to changing environment and stress such as antibiotics, pollutants, and heavy metals.
Within the matrix of biofilms, cells attain dormancy and undergo several phenotypic
changes including a subpopulation of non-replicating cells called persisters. Persister
cells are endowed with higher drug tolerance and can survive nearly 1000-fold
higher minimum inhibitory concentration of drugs. The vast diversity of mechanism
for biofilm formation among different species of bacteria has posed a serious
challenge in terms of devising strategies to combat biofilm. A recent study has
implicated the role of chaperonic proteins in biofilm formation. Chaperons aid in
protein folding and shape the three-dimensional structure of many proteins simulta-
neously. Peptidyl-prolyl isomerases (PPIase) are a class of cyclophilins that are
ubiquitously expressed in prokaryotes and eukaryotes and are involved in
interconversion of cis and trans isomers of peptide bonds with proline residue.
PPIase possess chaperonic activity and regulate several key functions in bacteria
including iron regulation, signaling, and immune defense. In Mycobacterium tuber-
culosis, PPIase is present in two forms – PpiA and PpiB. M. tuberculosis PpiA share
structural and phylogenetic similarity with cyclophilins present in eukaryotes.
M. tuberculosis PpiB is essential for the survival of the pathogen and is involved
in biofilm formation (Pandey et al. 2016, 2017). M. smegmatis overexpressing
M. tuberculosis PpiB showed enhanced biofilm formation and exhibits drug toler-
ance to anti-TB drugs. As a result, higher dosage of anti-TB drugs are required for
treatment of patients suffering from tuberculosis (TB), thereby causing toxicity in
patients. The involvement of M. tuberculosis PpiB in biofilm formation as well as in
several key pathways of the bacterial metabolism makes it a “choke point” enzyme
for inhibitors. It has been shown that cyclophilin inhibitors such as cyclosporine A
and several FDA-approved drugs including acarbose can bind with PpiB.
Cyclosporine A, acarbose, and gallium nanoparticle can inhibit the activity of
M. tuberculosis PpiB, thereby suppressing formation of biofilm. The reduction in
biofilm formation results in higher penetrance of drugs through biofilm matrix.
Tackling biofilm-related infection is a cumbersome task due to the fact that several
heterogenous species of bacteria exist within biofilms. The use of inhibitors can
selectively eliminate a group of bacteria from the heterogenous population; however
a subpopulation of bacteria that are resilient to the activity of these inhibitors can
transfer gene to other bacteria. As a result, several inhibitors that have been tested
against biofilm-forming microorganism either failed or their efficacy gradually
reduced with time. The bottleneck in devising strategy for targeting biofilms has
been the lack of a target that is essential across a wide spectrum of microorganism.
PpiB can prove to be a novel target for biofilm-forming organisms due to its presence
in all biofilm-forming bacteria. Besides, PpiB homologues in several biofilm-
forming bacteria possess similar amino residues in the domain that binds with
cyclosporine A, acarbose, and gallium nanoparticle (Kumar et al. 2019). Targeting
PpiB could prove to be the masterstroke against a wide genre of biofilm-forming
bacteria involved in multiple pathogenic symptoms.
18.9 Pathology Associated with Biofilm-Forming Bacteria
Biofilms are a major reason behind the recalcitrant nature of pathogenic microbes
toward antimicrobial agents and host immune responses, as biofilms act as a physical
barrier against drugs and provide a protective niche for mycobacterial survival.
Conventional biofilms are extremely resistant to host inflicted insults and can endure
antimicrobial agents as much as 10–1000 times of that required to eradicate plank-
tonic microbes. Biofilms have also been established as a crucial factor in the
pathogenesis of a large number of bacteria. The nature of biofilms also makes it
formidable for the host immune cells to attack or opsonize bacteria harbored within
them (Piatek et al. 2013). S. aureus evades opsonization by phagocytic cells in blood
by producing coagulase that activates prothrombin and promotes production of
326 A. Alam et al.
insoluble fibrin which eventually leads to strengthening of the biofilm (Cheng et al.
2010). In cystic fibrosis patients, alginate and rhamnolipid production is enhanced
by P. aeruginosa as a strategy to escape macrophages and stimulate necrosis of
polymorphonuclear leukocytes, respectively (Van Gennip et al. 2009).
The oral cavity provides an excellent niche for survival of nearly 800 species of
microorganisms that form a complex-multispecies biofilm or plaque. Anaerobic
bacteria such as Porphyromonas gingivalis colonize the biofilm at a later stage,
erode the dental surface with acidic toxins, and cause periodontitis and peri-
implantitis. P. gingivalis differentially express nearly 20% of its genome during
the various stages of biofilm formation and virulence. The versatility of P. gingivalis
in attaching to oral soft tissue, dental implant, and other oral bacteria is due to the
presence of fimbriae on the cell surface. Loss of Fim A fimbriae results in inhibition
of adherence to epithelial cells and dental fibroblasts. Recent studies showed that
arginine plays a key role in fimbriae-mediated biofilm formation. The activity of
streptococcal Arc A, which catalyzes hydrolysis of arginine to citrulline, suppresses
fim A expression in P. gingivalis and prevents biofilm formation. Unlike other
bacteria that utilize siderophores for sequestration of iron, P. gingivalis possess
proteases such as gingipains for acquiring iron from host heme. Several species of
dental microbiota exhibit mutualistic coexistence with P. gingivalis. Isobutyric acid
produced by P. gingivalis aid in growth of Treponema denticola, while succinate
produced by T. denticola enhances growth of P. gingivalis. P. gingivalis infections
lead to chronic inflammatory disease of oral cavity characterized by loss of teeth and
increased risk of cardiovascular diseases.
M. tuberculosis is endowed with persistence and drug tolerance, typical of
biofilm-associated infections caused by pathogenic bacteria. It has been reported
that M. tuberculosis develops biofilms within host cells that allow it to evade the
immune surveillance. M. tuberculosis biofilms are associated with several other
non-tubercular bacteria and opportunistic pathogens. M. tuberculosis biofilms are
mainly composed of methoxy-free mycolic acids and its biosynthesis is modulated
by fatty acid synthase complex II-GroEL-mediated pathway. Disruption of polyke-
tide synthases, pks16 and pks1/15, is associated with reduced biofilm formation in
M. tuberculosis and suggests the role of surface lipids in initial aggregation of
mycobacteria. As such the high lipid content of the bacterial cell wall limits the
diffusion of drugs; biofilm-encapsulated mycobacterial cells show enhanced drug
refractory physiology. M. tuberculosis exhibit enhanced tolerance to up to 1000
times higher minimum inhibitory concentration of drugs due to the presence of
persister cells within the biofilms. S. epidermidis biofilm provides resistance to
phagocytosis by interfering with the activation of complement protein C3b (Kristian
et al. 2008). Biofilm infections have also been implicated in various other diseases
such as endocarditis, chronic otitis media, obstructive pulmonary diseases, and
chronic (diabetes) wound infections. Biofilms also dwell in patients with internal
medical devices for treatment (Table 18.1). Similarly, lung infections by
P. aeruginosa can be significantly attenuated by targeting the quorum sensing
pathway (O’Loughlin et al. 2013). Staphylococcal biofilm formation is demonstrated
to be suppressed in vivo by using RNAIII-inhibiting peptide.
Table 18.1 List of clinically relevant biofilm-associated diseases

Organ/
body part Drugs/
affected Organism Disease/symptom interventions
Eye Staphylococcus epidermidis, Endophthalmitis, Tobramycin,
(contact Staphylococcus aureus, keratitis, scleral buckle polymyxin,
lens) Staphylococcus infection, lacrimal bacitracin,
saprophyticus, Serratia infection, periorbital levofloxacin
marcescens, Fusarium infections
solani
Wound Bacillus, Clostridium, PASH syndrome, delayed Infliximab,
(burns) Staphylococcus epidermidis, healing in burns clindamycin,
Staphylococcus aureus metronidazole,
Amikacin
Dental Porphyromonas gingivalis, Dental plaque, dental Sodium
Lactobacillus casei, caries, pulpitis, apical hypochlorite,
Streptococcus sobrinus periodontitis, gingivitis acetaminophen,
corticosteroid
Prosthetic Enterococci, diphtheroids, Endocarditis Ampicillin,
heart valve candida spp. gentamicin,
penicillin G
Breast Staphylococcus epidermidis, Chronic infection of Chloramex,
implant Corynebacterium, breast Fucidin,
Mycobacterium fortuitum Terramycin-coated
implant
Urinary Neisseria gonorrhoeae, Urinary tract infections Fluoroquinolone,
catheter Staphylococcus aureus, cephalosporin,
Enterococcus faecalis aminoglycoside
Orthopedic Pseudomonas, streptococci, Chronic osteomyelitis, Titanium nitride
metal Proteus mirabilis peri-prosthetic joint coating,
implants infections gentamicin-coated
polyurethane
sleeves
Lung Burkholderia cepacia, Cystic fibrosis, Ivacaftor,
Pseudomonas aeruginosa, ventilator-associated bronchodilators,
Aspergillus fumigatus pneumonia ureidopenicillin,
carbapenem
Ear Haemophilus influenza Influenza, chronic otitis Ofloxacin ear drop,
media amoxicillin
Vagina Corynebacterium spp., Intrauterine device- Metronidazole,
Micrococcus spp., Candida associated infections, gentamycin
albicans, Lactobacillus Gardnerella vaginitis
plantarum, group B
streptococci
328 A. Alam et al.
18.10 Anti-biofilm Strategies
Due to the adverse impact of biofilms on human health, various methods such as
mechanical disruption, disinfectants, chlorination, and ultraviolet have been used
traditionally. Recent advances in mechanism of biofilms have provided new insights
to control biofilm-based infections. Table 18.2 shows the list of various anti-biofilm
strategies that are either used alone or in combination. Extracts from plants such as
Rhodiola crenulata, Malus pumila, Dolichos lablab, etc., show potent anti-biofilm
activity. Chemical derivatives of active ingredients of plant extracts such as icariin
and resveratrol can be synthesized and exhibit significant anti-biofilm activity. Green
tea possesses high level of anti-oxidants and can suppress biofilm caused by
Streptococcus mutans. Honey possesses antimicrobial property due to the presence
of antimicrobial peptide, defensins, and is effective in inhibiting Enterococcus spp.
biofilm formation. Similarly, several essential oils such as cumin oil, cinnamon oil,
and oregano oil are effective against biofilm caused by Klebsiella pneumonia and
mixed-species biofilms of enteropathogenic E. coli and S. aureus, respectively (Ong
et al. 2018). The emergence of antibiotic resistance has highlighted the use of
bacteriophages in controlling bacterial infections. Phages are highly specific toward
bacterial targets, persist as long as the host bacterium is present, and do not show side
effects on normal microbiota. Bacteriophages can potentially release enzymes such
as polysaccharide depolymerase that liquefy the biofilm matrix and allow the phage
to penetrate the biofilm. Genetically engineered phages such as E. coli-specific
phage (T7), cloned with dispersin (dspB) gene, have shown higher efficacy in
biofilm degradation. The major technological challenges in using phages for anti-
biofilm strategy are i) reducing the release of toxins by phage, ii) overcoming
bacterial resistance to phages, and iii) neutralizing the virulence genes of phages
from incorporating into the host bacterial genome. The quorum sensing
(QS) molecules such as acetyl homoserine lactone (AHL), autoinducer 2 (AI-2),
etc., aid in aggregation of bacteria. Studies have shown that inhibitors that quench
the activity of quorum sensing molecules can disrupt the aggregation of cells,
considered as an essential step in biofilm life cycle. Naturally occurring quorum
sensing inhibitors such as furanone, ajoene, naringin, curcumin, epigallocatechin
gallate, etc., have shown anti-biofilm properties against several gram-negative and
gram-positive bacterial species. Quorum quenching enzymes such as AHL
lactonases, AHL acylases, and oxidoreductases are effective in disruption of biofilm
without affecting the microorganism. Therefore treatment with antibiotics in combi-
nation with QS molecules or quorum quenching enzymes can be effective in
targeting bacteria within biofilms. QS inhibitors such as furanone and penicillic
acid increased the efficacy of antibiotic tobramycin against P. aeruginosa biofilms.
Blockage of cell to cell communication by using peptides such as RNA III-inhibiting
peptide (RIP) that inhibits phosphorylation of TRAP (target of RNA III) proteins can
prevent initial stages of biofilm formation. The formation of biofilms on medical
devices and implants poses a unique challenge for engineering devices that are inert
Table 18.2 List of anti-biofilm strategies

Strategy Mode of action Efficacy against
Plant extracts Act as antioxidants Streptococcus mutans,
Green tea, Lactobacillus plantarum,
Cuminum S. epidermidis
cyminum
Cinnamon oil
Oregano oil
Thyme oil
Bacteriophage Infects and lyse bacteria cells P. aeruginosa, P. fluorescens
Engineered with
dispersion B gene
Phage PhilBB-PF
7A
Phage in
combination with
antibiotics
Quorum sensing Blocks intercellular communication P. aeruginosa, B. cepacia,
inhibitors carried out through quorum sensing S. aureus, Y. enterocolitica
AHL cyclase molecules
AHL lactonases
AIP inhibitors
Oxidoreductases
Surface Prevents attachment and aggregation S. epidermidis, biofilms on heart
modification of cells on surface devices
Coating with
furanone
Zirconium oxide
Nanocrystalline
silicon carbide
Polyethylene
glycol
Nanoparticle Generation of reactive oxygen Candida spp., E. coli,
Silver species, destabilizing cell membrane Enterococcus spp., Salmonella
Gallium nitride and enzymes spp.
Titanium oxide
Silica
nanoparticle
Enzymes Breakdown of components of biofilm Streptococcus pyogenes,
DNase 1 matrix Acinetobacter baumannii,
Lysostaphin Haemophilus influenza
Lyase
Photodynamic Activation of photosensitizing drugs C. albicans, Enterococcus
Therapy due to light faecalis
Methylene blue
Toluidine blue
(continued)
330 A. Alam et al.

Strategy Mode of action Efficacy against
Bacteriocin Formation of pores on cell membrane Listeria monocytogenes,
Nisin A of bacteria S. aureus
Lacticin Q
Ultrasonic Mechanical disruption E. coli, P. aeruginosa
treatment
>20 kHz
frequency of
sound waves
Anti-biofilm Disruption of initial stages of biofilm Wound colonizing P. aeruginosa,
agents matrix peridontal colonizing
Molsidomine Aggregatibacter
Diethylamine actinomycetemcomitans,
nonoate Salmonella spp., Porphyromonas
diethylammonium gingivalis
Povidone iodine
Xylitol
Dispersin B
to biofilm formation (Koo et al. 2017). Since surface attachment is the first step of
biofilm formation, coating the surface with inert substances or chemical modification
of surface is an innovative strategy for preventing biofilm formation on medical
devices. Surface adsorption of furanone and quaternary ammonium salts prevents
Staphylococcus epidermidis biofilm formation on catheters. Nanoparticles such as
silver possess antimicrobial properties. Surface medication with silver coating
prevents biofilm formation and studies have shown that silver nanoparticles can
reduce 97% of biofilm formation caused by Candida spp. Similarly, titanium dioxide
nanomaterials are effective against fungal biofilms and are widely used in dental
implants (Roy et al. 2018). Gallium shares similar charge and shape with ferric ion
and binds with iron siderophores. Iron acts as cofactor of key enzymes involved in
bacterial metabolism and disruption of iron metabolism can significantly inhibit
growth of bacteria. Gallium competes with ferric ion and inactivates Fe
siderophores, thereby leading to suppression of growth and metabolism of bacteria.
Recent studies show that gallium nanoparticles not only inhibit biofilm formation
but also improve the efficacy of anti-tuberculosis drugs against mycobacterial
biofilms (Kumar et al. 2019). Several constituents of biofilms such as DNA, proteins,
and polysaccharides can be targeted by using enzymes. A mixture of
deoxyribonucleotides, glycosidases, and proteases has shown significant anti-
biofilm potential. Treatment with DNase1 (5 μg/ml) either alone or in combination
with antibiotics (rifampicin, ampicillin, azithromycin, etc.) reduced biofilm forma-
tion of S. aureus, S. pyogenes, Acinetobacter baumannii, Haemophilus influenza,
etc. Lysostaphin (LS) in combination with nafcillin and enzymes such as amylase,
lyase, and lactonase are effective against methicillin-resistant S. aureus (MRSA).
Photodynamic therapy is the process of activating drugs, called photosensitizer,

using light of particular wavelength. During the exposure of light, photosensitive
drugs produce reactive oxygen that destroys adjacent cells. Oral biofilms that consist
of multispecies microorganism are treated with helium laser light in the presence of
toluidine blue.
18.11 Conclusion
Combating microbial biofilm infections and getting rid of it completely is a daunting

task for clinicians. Tackling biofilms is difficult as there are several proteins that are
involved in biofilm formation and the involvement of these proteins also varies
across biofilm-forming organism. Prolonged treatment with combinations of
antibiotics that possess different killing mechanisms can be used to eliminate the
infection. However, the efficacy of this method may still be questionable. The
mutation rate of bacterial genome is high and offers it a unique advantage to adapt
to stress. As a result of mutation the protein targets change their conformation or the
active site amino acids get altered, thereby reducing the interaction of inhibitors to
target genes or proteins. Apart from this genotypic adaptation, bacteria can tempo-
rarily adapt phenotypically by altering the secretion of extracellular molecules that in
turn form an impregnable coating. The extracellular coating or matrix can deposit
over biotic or abiotic surfaces. In other cases, removal of medical devices and
abscesses is necessary for a successful outcome. Treatment only with antibiotics is
not a viable option in the case of biofilm-associated infection, partly because the
mutation in bacterial genes renders the protein targets inaccessible to drugs and the
gradient of drugs across the matrix decreases toward the core of the biofilm.
A successful medicament against biofilm would involve a combination of several
anti-biofilm strategies that target different steps of biofilm formation. In the light of
the fact that dissemination of persister cells from the mature biofilm is preceded by
dissolution or loosening of the matrix, the auto-dissolution of matrix is governed by
mechanisms that are inherently controlled by the persister cells. The signal for stress
removal is communicated from the cells on the outer surface of the matrix to the
persister cells within the core of the matrix. It may inevitably lead to change in gene
expression of persister cells that in turn result in generation of “factors” that
eventually dissolve the matrix. RNA sequencing of persister cells may shed light
about “wake up” genes that regulate the expression of these “factors” that can
potentially dissolve the matrix. The usefulness of these “factors” as a futuristic
tool against biofilm-associated infections is an open question. Other future strategies
for biofilm control may include targeting the essential regulatory pathways such as
nucleotide signaling and damaging the amyloid structures which play a key role in
biofilm formation and adaptation (Romero et al. 2010). Alternatively, chaperonic
PpiB proteins, considered as a “choke point protein” involved in biofilm formation
across many biofilm-forming pathogens and which regulate other proteins in the cell,
could pave the way for a successful target against biofilm-borne infections.
332 A. Alam et al.
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Part III
Vaccines, Diagnostics and New Interventions
Best Practices in Mycobacterial Research
Laboratories 19
Noman Siddiqi
Abstract
Research on Mycobacterium tuberculosis (MTB), the bacterium causing tubercu-
losis (TB), has seen a significant increase in the last decade. Advances in
molecular biology techniques now provide scientists with a plethora of
approaches to investigate bacterial pathogenesis in the laboratory and in animal
models. Here we outline some good laboratory practices for working with MTB
in a research setting. The risk of bacterial transmission via aerosols or through
accidental exposure requires that we implement rigorous practices to mitigate this
risk. Most laboratory research on MTB is performed in specialized laboratories at
biological safety level 3. We discuss primary and secondary barriers to MTB
exposure (infection), controls that reduce the risk of exposure to the bacterium
during experimental manipulation, provide guidelines for the prudent use of
biosafety cabinets and personal protective equipment, and present examples of
standard operating protocols, including setup of biosafety cabinets, waste man-
agement practices, equipment use, management of spills, occupational health
programs, and facility decontamination.
Keywords
Mycobacterium tuberculosis · BSL3 · Respirators · Tuberculosis · Risk
assessment · PPE · Laboratory design · Engineering control · Occupational
health · Bacterial inactivation · Spill cleanup
N. Siddiqi (*)
Harvard TH Chan School of Public Health, Boston, MA, USA
e-mail: nsiddiqi@hsph.harvard.edu

https://doi.org/10.1007/978-981-32-9413-4_19
338 N. Siddiqi
Abbreviations
A/BSL3 Animal biological safety level 3

BI Biological indicators
BSC Biosafety cabinet
BSL1 Biological safety level 1
DPM Differential pressure monitors
HEPA High-efficiency particulate air
IGRA Interferon -gamma release assay
OH Occupational health
PAPR Powered air-purifying respirators
PCR Polymerase chain reaction
PPE Personal protective equipment
RT-PCR Reverse transcription polymerase chain reaction
SOP Standard operating procedure
TB Tuberculosis
VHP Vapor Hydrogen Peroxide
19.1 Introduction
In the past two decades, mycobacterial research has seen immense growth (Bloom
et al. 2017). With the development of tools to target mycobacterium at the genetic
level (Hatfull and Jacob Jr 2014), laboratory-based research has expanded and
flourished, globally. As molecular biological technologies have proliferated, so has
our understanding of the risks associated with performing research on this pathogen.
Accordingly, biological safety considerations have evolved over the years, to ensure
science is conducted in a safe environment (Fleming 2006; Wooley and Byers 2017).
This has led to a transformation in research practices, biosafety program manage-
ment, and risk assessment (World Health Organization 2012). While researchers
have innovated in the laboratory, architects and engineers have worked on improv-
ing laboratory systems and building design. Likewise, equipment manufacturers
have addressed safety concerns by improving design and safety features. However,
although most institutions and governments have instituted policies to regulate the
operation of high containment laboratories, there is still a need to define global
operating standards for such laboratories and work practices.
A discussion on the risks associated with manipulating Mycobacterium tubercu-
losis (MTB) and risk mitigation strategies in research laboratories is presented
below.
19 Best Practices in Mycobacterial Research Laboratories 339
19.2 Risk Assessment
The following factors are generally considered when performing risk assessment
(Fleming 2006):
1. Characteristics of the agent/material (Fig. 19.1).

2. Activities that may lead to exposure (Fig. 19.2).
3. Potential of the exposure to cause infection or injury.
4. Consequences of the ensuing infection or injury.
Fig. 19.1 Characteristics of Mycobacterium tuberculosis
Fig. 19.2 Activities leading to exposure

340 N. Siddiqi
Mycobacterium tuberculosis (MTB) is a nonmotile bacterium, typically transmit-

ted via aerosols (Cole et al. 2005; Pfyffer 2007). Figure 19.1 highlights some salient
characteristics of the bacterium (Public Health Agency of Canada 2010; Hirai 1991;
Kramer et al. 2006; Riley and Nardell 1989). Figure 19.2 describes activities routinely
performed in a research setting, involving manipulation of MTB. The site and level of
exposure contributes to the establishment of a potential infection. Whether this
infection leads to disease is dependent on the health status of the individual, on
whether prophylactics (e.g., vaccines) were administered, and whether treatment was
received following infection. Host factors such as age, immune status, immunodefi-
ciency, etc., also contribute to the clinical disease outcome (Bloom et al. 2017).
Based on their risk assessment, pathogens are classified into distinct risk groups
and their research falls under specific laboratory designations (World Health Orga-
nization 2004; Centers for Disease Control and Prevention 2009) (Fig. 19.3).
Members of the Mycobacterium tuberculosis complex, including M. bovis,
M. africanum, M. microti, M. canetti, M. caprae, and M. pinnipedii, are considered
risk group 3 pathogens and are usually assigned a biological safety level 3 (BSL3)
laboratory designation, for research purposes. The Biosafety in Microbiological and
Biomedical Laboratories (BMBL) definition for biological safety level 3 (BSL3),
“. . .is applicable to clinical, diagnostic, teaching, research, or production facilities
where work is performed with indigenous or exotic agents that may cause serious or
potentially lethal disease through inhalation route exposure”. Laboratory personnel
must receive specific training in handling pathogenic and potentially lethal agents, and
must be supervised by scientists competent in handling infectious agents and
associated procedures. All procedures involving the manipulation of infectious
materials must be conducted within Biosafety Cabinets (BSC), other physical con-
tainment devices, or by personnel wearing appropriate personal protective equipment.
A BSL-3 laboratory has special engineering and design features.” (Centers for Disease
Control and Prevention 2009).
19.3 Risk Control
Risk mitigation strategies can be divided into two groups – primary and secondary
barriers of control.
Primary barriers provide protection to the researcher and include equipment like
biosafety cabinets (BSC) and personal protective equipment (PPE).
Secondary barriers encompass engineering and administrative controls, which
provide protection to the facility users and the surrounding environment. Engineer-
ing controls cover laboratory design, equipment containment, and placement, while
administrative controls include the training requirements, biosafety program, occu-
pational health requirements, standard operating procedures (SOP), and protocol risk
assessment.
Fig. 19.3 Risk group definition
19.4 Biosafety Cabinet
A laminar flow biological safety cabinet (BSC) provides three kinds of protection: a)
protection to personnel, b) protection to the product, and c) protection to the
environment. A basic understanding of BSC operation is essential for researchers
working in a BSL3 facility. A common mistake includes covering the air intake grills
342 N. Siddiqi
Fig. 19.4 Different types of biosafety cabinets. (Source: Baker Company)
in the front and back of the BSC; this blocks the air flow, compromising the aseptic
environment. Other operational errors include placing the BSC under supply or
exhaust air ducts, next to entry doors, overcrowding the cabinet with equipment/
samples, and simultaneous use of the cabinet by multiple researchers.
BSCs are grouped into three classes based on their functionality (Fig. 19.4).
BSC class II type A2 cabinets are the most commonly used types in the research
laboratory. When working with large amounts of volatile chemicals, it is preferable
to use type B cabinets. Class III cabinets offer the highest levels of protection and
containment.
19.5 Respirators
Although different classes of respirators are available, not all provide protection
suitable for an MTB research laboratory. The two most favored respirators for MTB
research are N-95 and powered air-purifying respirators (PAPR).
N-95 respirators are one of the more frequently used respirators. Researchers get
fit tested for the make and model of the respirator. Ideally, the fit test is repeated
annually, as the fit of the mask is molded to the contours of the face (NIOSH 2013).
Facial hair – even a stubble – can interfere with the efficiency of the respirator.
PAPRs use air displacement from the wearer’s mask to provide protection. This
involves a battery-operated motor blowing filtered air into the mask, which could be
a partial- or full-face mask (Roberts 2014).
Individual respirators provide varying degrees of protection from aerosolized
contaminants. Usually a PAPR with a full-face mask offers better protection than
Fig. 19.5 Personal protective equipment (PPE)
an N-95 respirator. A PAPR with a full-face mask is recommended for experiments

involving a high risk of aerosolization of the pathogen, such as flow cytometry,
infection, or homogenization. The PAPR airflow is battery-powered, so it is critical
to confirm the battery is adequately charged before use.
19.6 Other PPEs
To prevent contamination of clothes it is best to wear a Tyvek jumpsuit (over street

clothes) when working in the BSL3, while other individual protective coverings
consisting of disposable gowns, hoodies, booties, and long sleeves can also be used.
Some laboratories require users to change into scrubs and then wear a Tyvek suit. It
is also important to follow the “two pairs of gloves” rule when handling the
pathogen. Extra Tyvek sleeves and booties (shoe covers; see Fig. 19.5) could be
worn to provide extra protection to arms and feet, as these are more likely to get
contaminated during work. Figure 19.6 outlines the steps for wearing and removing
PPE while entering or exiting the BSL3 laboratory.
19.7 Engineering Control – Design of the Laboratory
The BSL3 laboratory builds on features present in BSL1 and BSL2 laboratories.
Specifically, it has a specialized ventilation system allowing for directional airflow,
airtight seals and finishes, access controls, waste management, and many other
344 N. Siddiqi
Fig. 19.6 (A&B): Typical donning and doffing procedure for PPE
features. Figure 19.7 illustrates some recommendations for laboratory design, drawn
from the NIH handbook (Memarzadeh and Wheeland 2016).
Directional airflow is set up to allow clean air to enter the facility and to exit
through exhaust ducts with high-efficiency particulate air (HEPA) filters. This
prevents any contaminated air from escaping the laboratory into adjoining clean
areas. There is no recirculation of air in these laboratories. It is best to have a
Fig. 19.7 Laboratory design recommendations
dedicated air supply and exhaust system serving the BSL3. These systems should be
hardwired so that the supply fan automatically shuts down if the exhaust fan fails,
preventing the space from “going positive,” i.e., allowing air to flow out of the
facility. Differential pressure monitors (DPM) should be installed for each door and
wired to an alarm (visual and audible) to indicate a failure in the system. Researchers
need to be trained to monitor the DPMs and exit the laboratory if the alarms turn
on. Installing redundant HEPA filter units on the exhaust system will allow for easy
maintenance of the filters. The air supply handler and exhaust fan systems supplying
the BSL3 are critical for the safe operation of the laboratory and should have a
backup power source.
BSCs and autoclaves are essential to the functioning of the BSL3 laboratory.
Ideally, a pass-through autoclave, with one door opening inside the laboratory and
another outside, should be installed. This allows for loading of contaminated waste
inside the laboratory. Once the sterilization cycle is complete, the autoclave door
outside the laboratory can then be used to remove waste. It is best practice to confirm
the sterilization performance of the autoclave periodically (Association for the
Advancement of Medical Instrumentation 2017), e.g., by running challenge loads
with biological indicators (BI) every fortnight.
346 N. Siddiqi
19.8 Waste Management
It is important to set up procedures for handling waste generated in the laboratory.

Waste streams are defined based on methods of disinfection and/or sterilization.
Broadly, waste can be classified into two streams: either created inside the biological
safety cabinet or outside it. Waste generated within the BSC, being potentially more
contaminated and with a higher risk of personnel exposure, needs to be disinfected
and sterilized, while waste/trash generated outside the BSC carries less risk and may
be treated less stringently. One should add multiple steps of disinfection and a final
sterilization cycle for waste generated inside the BSC. Our laboratory typically uses
chemical disinfection procedures to treat waste inside the BSC before it is bought out
and autoclaved for sterilization. Waste generated outside the cabinet is packed and
autoclaved without undergoing a chemical disinfection process first.
The cabinet waste stream can include solid, liquid, or sharps waste, each of which
needs to be processed differently. One practice is to pack solid waste in a single
biohazard bag, liquids in plastic containers or bottles, and sharps in approved
cardboard or plastic containers. Consider adding disinfectant at a defined concentra-
tion to the liquid waste, to allow for initial disinfection. Sharps can be dipped in
disinfectant before being discarded in approved sharp containers. Solids can be
wiped with disinfectant and packed in a bag.
All waste generated in the laboratory should be sterilized before being removed
from the laboratory. It is recommended to have a double door autoclave to facilitate
this process. The double door allows the user to define a “dirty” and “clean” side in
the laboratory. Each sterilization run is typically monitored by chemical indicators
and built-in sensors in the autoclave which measure pressure, temperature, and
exposure time (Association for the Advancement of Medical Instrumentation 2017).
19.9 Occupational Health, Disease Surveillance, and Response

to Exposure
An occupational health (OH) program can cover (1) prejob offer and annual health
screening, (2) annual fit test for N-95 respirators, (3) identification of high-risk
individuals (e.g., immune suppressed or pregnant), and (4) annual updates of an
exposure control plan.
Annual health screens help in disease surveillance and can potentially identify
unreported or unidentified exposure. This can help in re-evaluating risk associated
with specific procedures and suggest PPE or engineering controls to mitigate the risk
of exposure.
Occupational health providers can also provide information to researchers on host
factors which influence infection and clinical features of tuberculosis (TB). Some of
these include age, vaccination (BCG) status, immune/immunodeficiency status,
coexisting diseases, and drug treatments.
If potential exposure is suspected, contacting the occupational health provider
immediately and following their recommendations is critical. Many laboratories
keep pathogen information cards which can be presented to health providers by the
researcher after potential exposure in the laboratory. These cards list information
about the pathogen, disease, and potential testing and treatment options. Diagnostic
testing may include the tuberculin skin test (TST) or the interferon gamma release
assay (IGRA) test for determining a basal value of MTB antigen-specific immune
response (Mazurek et al. 2007; Kimura et al. 1999). This is then used for evaluating
infection progression following subsequent testing and for recommending treatment
options.
19.10 Administrative Controls – Training Requirement
An important way to reduce incidents and exposure in the laboratory is to train users
in best laboratory practices. A combination of classroom teaching and hands-on
training is recommended. As an example, we require all our researchers to undertake
15–20 h of hands-on training and 1 h of didactic training. Beyond this, our
researchers also get a chance to shadow other scientists working in the BSL3.
Annual training refreshers reinforce various biosafety practices and correct use of
equipment in the BSL3.
The hands-on training module is designed to simulate a typical experiment,
including BSC setup, culturing bacteria (in liquid and solid media), waste manage-
ment, dealing with spills, a potential exposure situation, decontamination
procedures, and cleaning of the BSC post-experiment. These are offered as individ-
ual sessions with expert trainers. After three to four rounds (at least 3 h each) of such
sessions, the trainee is required to demonstrate their understanding and ability to
work independently in the BSL3, as judged by their peers, who have experienced
working in the BSL3, to confirm their readiness to work unsupervised in the
laboratory. Investigators are required to undertake similar training for any new
procedure that they need to perform in the laboratory.
BSL3 users participate in reviewing protocols and procedures before they are
implemented in the laboratory. This ensures that all users are familiar with the
protocols and associated risks. The group vetting of procedures also allows us to
maintain stringent safety standards across the gamut of experimental procedures.
19.10.1 Access
Once a trainee has met all training requirements they are allowed access to the
laboratory. Users are encouraged to work in pairs when working off-hours and
during holidays. All entry/exit doors into BSL3 should have card access control.
Researchers with access are trained to not share their access cards and to bar visitors
or untrained people from the laboratory.
Casual visitors should not be allowed into the biocontainment facility. When
scientifically necessary or approved, untrained individuals may enter the facility
only when accompanied by an approved user. This is especially important for
tradesmen and engineers who need to enter the facility to perform repairs.
348 N. Siddiqi
19.11 Standard Practices and Precautions
The majority of work practices for BSL3 builds on practices and precautions for
BSL2 laboratories (Emmert 2013). Additional practices are listed below:
• Stored items should be packed correctly and clearly labeled with investigator’s
name, date, and content information.
• When bringing hands out of the BSC:
– Wipe gloves with disinfectant.
– Discard the second pair (outer pair) of gloves.
– Remove hands from cabinet without touching anything.
– Wear a new pair of gloves before carrying out further procedure.
• All vessels containing pathogens should rest on the floor of the BSC while
transferring cultures from one container to other to avoid any spillages or splashes
as a result of these containers slipping out of the hands.
• To bring items out of the BSC during an experiment/procedure, a space should be
identified as “clean.” A surface would be considered clean only after it is wiped
down with disinfectant.
• Before an item is removed from the cabinet it should be wiped (surface decon-
tamination) and placed on the clean side.
• Any manipulation of a pathogen in the BSC is considered to render the BSC
“dirty,” necessitating the need to clean a side before removing items from
the BSC.
• Ideally, all pathogen manipulation should occur only in the BSC. If this is not
possible, a risk assessment should be performed and steps to minimize risk
implemented. An example of minimizing aerosol risk during centrifugation is
to use aerosol caps on rotor buckets.
19.12 Use of the Biosafety Cabinet
The biosafety cabinet is part of the primary barrier and offers protection to users and
the environment when correctly set up and used. Generally, BSCs in the BSL3
should be tested and certified every 6 months.
• Ensure that start-up and purge cycles of BSC are properly functioning and BSC
was disinfected by previous user.
• The BSC floor can be lined with an absorbent sheet (to contain liquid spills) and is
removed at the end of each experiment as part of BSC cleanup.
• Containers/bags to capture different kinds of waste generated during the experi-
ment should be placed in the cabinet. Disinfecting wipes and a container of
disinfectant should be placed inside (Fig. 19.8). The BSC should be set up to
allow one side to be used for bringing items out (the “clean” side). Manipulations
Fig. 19.8 Biosafety cabinet setup for experiment
should occur on one side of the BSC, being careful to maintain a “clean to dirty/
contaminated” direction of work flow.
• Pathogen-containing items must always be in double containment (boxes/bags)
when taken outside of a BSC.
• Items must be cleaned/disinfected when moving from the dirty to the clean side of
the BSC.
• Objects must be cleaned/disinfected before being moved out of the BSC.
• A secondary container should be installed on the clean side to move liquid
cultures out of the BSC.
19.13 Exposure
Failure to wear the right PPE or breakdown of PPE may lead to exposure to the
pathogen. The risk is elevated when working with sharps and animals. Needlesticks,
cuts, animal bites, and scratches are potential exposure routes. In some instances,
PAPR failure or the N 95 mask coming off may expose the investigator. Rips and
nicks in primary and secondary gloves also pose a high risk of exposure. Equipment
failure, e.g., loss of power to the biosafety cabinet, spills in a centrifuge or
incubators, and sprays/splashes from a flow cytometer or plate reader could also
potentially lead to exposure.
The laboratory should have a plan in place to manage exposures to the investiga-
tor(s) or the environment. These plans should consider occupational health
350 N. Siddiqi
(OH) intervention for the exposed employee(s), containment of the material within
the lab, and steps to mitigate the risk associated with cleanup.
Following potential exposure, an investigator(s) should take the following steps:
• Exit the facility as per BSL3 SOPs.

• To get help, or if unable to exit, use the phone or panic alarm/distress button.
• Inform the principal investigator, laboratory director, or a colleague.
• Call the occupational health physician and follow their instructions.
• If directed by the physician or if unable to reach a healthcare provider, report to
the nearest hospital emergency room.
• Create a summary (if possible) of the incident, including information about the
pathogen, possible route of exposure, any drug resistance strain, OH physician’s
name, and phone number.
19.14 Inactivation of Bacteria for Sample Removal
Occasionally, researchers may need to remove samples from the BSL3 laboratory to
a BSL2 laboratory to conduct follow-on experiments; e.g., bacterial samples might
need to be processed for nucleic acid/protein/metabolite analysis or cell lines may be
removed for downstream experiments involving PCR, RT-PCR, proteomics,
cytometry, or mass spectrometry.
Before removal, samples need to be processed to clear them of any live bacteria.
We can use physical or chemical means to sterilize samples. The choice of method is
determined largely by the downstream experiments to be performed on these
samples. Physical methods such as heat inactivation and filtration are widely used.
Chemical methods include the use of formalin, glutaraldehyde, paraformaldehyde,
phenol, methanol-chloroform, etc. (Schwebach et al. 2001; Doig et al. 2002).
Figure 19.9 describes some methods we use for transferring samples from BSL3
to BSL2. It is important that users validate each method that they intend to use for
bacterial inactivation. Validation would involve following the exact steps in a
planned experiment, exposing the sample to the inactivation reagent, washing the
sample, and plating it to check for bacterial growth. A strict standard applies here:
there should be no bacterial growth on the plates after 6 weeks of incubation.
Positive and negative controls should be included in this testing.
19.15 Decontamination
Various methods are used for decontaminating work surfaces in the laboratory. An
adequate concentration of disinfectant and sufficient exposure time are important
factors that determine the efficacy of decontamination. Certain surfaces may be
Fig. 19.9 Commonly employed inactivation methods
harder to decontaminate than others (e.g., porous versus nonporous material) may.
Surface decontamination can be performed using disinfectants (wipe down). Newer
methods of decontamination, such as the use of vapor hydrogen peroxide (VHP)
(Hall et al. 2007) and chlorine dioxide (Lowe et al. 2012), are very effective for
decontaminating large areas, such as rooms and suites. However, these methods
require specialized equipment and trained professionals, unlike surface wipe down
methods, which can be done by laboratory staff. One also needs to verify there are no
leaks from the laboratory before using VHP or chlorine dioxide. The choice of one
method over another is based on risk assessment and biosafety considerations.
Equipment in need of repair (or being disposed of) can be removed from the
laboratory after gaseous decontamination. Smaller equipment can be
decontaminated inside a BSC, while larger pieces need to be isolated, either in a
room or covered by a flexible sheet (plastic tent), for decontamination.
19.16 Spills and Cleanup
Strict precautions should be taken when working, transporting, or removing samples

containing MTB. Spills can occur when researchers fail to follow safe practices as
outlined in the above sections. Spills can happen inside a biosafety cabinet when a
tube is dropped while pipetting samples. Spills can also occur outside of the BSC, in
a storage area, or within equipment (for example, tubes breaking in the centrifuge, a
culture flask breaking in the incubator shaker, or a leak in a primary container
containing MTB). Spills in the BSC are contained and so are easily dealt with,
unlike spills in equipment or general laboratory areas.
352 N. Siddiqi
Fig. 19.10 Protocol for cleaning a spill in centrifuge
Spills in the BSC can be cleaned with a disinfectant-soaked wipes or paper

towels. It is important to have an absorbent underpad covering the floor of the
BSC. These underpads can be replaced if a spill occurs. Always disinfect a spill
area first before continuing with the experiment.
Spills in equipment and general work areas need more intensive cleanup. Con-
sider isolating the area and evacuating the laboratory for an appropriate period of
time. The time will depend on the number of air changes needed in the space for
removal of any aerosolized particulates (Morbidity and Mortality Weekly Report
(MMWR) 2005). Discard any disposable PPE worn at the time of the spill and
decontaminate the rest before reuse. Wear adequate PPE and use respiratory protec-
tion which offers maximum protection (e.g., PAPR with a full-face hood), when
coming in to clean the spill. It is advisable to work in pairs when cleaning a spill. An
example of spill cleanup procedure in a centrifuge is shown in Fig. 19.10.
19.17 Summary
A BSL3 laboratory requires both engineering and administrative controls. A thor-

ough risk assessment should drive the setup of standard operating procedures (SOPs)
and experimental practices. The description of facility and practices outlined here are
based on risk assessment of Mycobacterium tuberculosis and its manipulation in a

research laboratory setting. Clinical laboratories may share some practices men-
tioned here but are typically not held to the same standards.
The emphasis on biological safety in research ensures that pathogens are handled
with utmost caution. The best practices outlined here ensure the prevention of
laboratory-acquired infections (Kao et al. 1997) or accidental release of the pathogen
to the environment. While some practices can be modified based on facility design or
equipment, others form the bedrock of MTB research. An example of the former
case would be the choice of PPE, which could be N95 or a PAPR. An example of the
latter case would be the inactivation of pathogen-containing samples before removal
from the BSL3 laboratory.
These practices should not be viewed as static but as requiring constant
re-evaluation and updating. Research procedures are evolving continuously and it
follows that we should likewise continually improve and adapt best practices for
handling mycobacterium in the laboratory.
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Clinical Aspects and Principles
of Management of Tuberculosis 20
Ashfaq Hasan, Sai Haranath Praveen, Chandrakant Tarke,
and Fahad Abdullah
Abstract
Tuberculosis over the ages, has killed more people than any other infection has.
Notwithstanding the advances in modern science, clinical diagnosis sometimes
remains elusive, owing principally to the frequent paucibacillary occurrence of
the disease and the slow doubling time of the organism; empiric treatment is often
fraught with risks in the era of increasing drug resistance.
This chapter attempts to provide an overview of the disease, beginning with the
pathogenesis and its protean clinical presentations. It also discusses the recent
evolution of molecular methods that have lately provided an impetus to early
diagnosis with a clear opportunity to unmask drug resistance before initiating
“blind”, potentially ineffective, and sometimes harmful treatment with standard
therapy.
The chapter also provides insight into tuberculosis in special situations, and
discusses briefly the treatments in uncomplicated cases as well as in special
situations, and in instances of drug resistance. Preventive methods including
current and upcoming vaccines are mentioned.
Finally, a short discussion of the sequelae of tuberculosis—which have the
potential to be confused with active disease—is presented.
Keywords
Pulmonary · Infection · Tuberculosis
A. Hasan (*) · F. Abdullah

Department of Respiratory Medicine, Deccan College of Medical Sciences, Hyderabad, India
e-mail: principal@deccancollegeofmedicalsciences.com
S. H. Praveen
Department of Pulmonary and Critical Care Medicine, Apollo Hospitals, Hyderabad, India
C. Tarke
Department of Pulmonary and Sleep Medicine, Apollo Hospitals, Hyderabad, India

https://doi.org/10.1007/978-981-32-9413-4_20
356 A. Hasan et al.
Abbreviations
ADA Adenosine deaminase

ADRs Adverse drug reactions (ADRs)
AFB Acid-fast bacilli
ARDS Acute respiratory distress syndrome
ART Antiretroviral therapy
ATT Anti-tubercular therapy
BAL Broncho-alveolar lavage
DOTS Directly observed treatment
EMB Ethambutol
GABA Gamma-aminobutyric acid
INH Isonicotinylhydrazide
IRIS Immune reconstitution syndrome
LAM Lipoarabinomannan
LPA Line probe assays
LTBI Latent TB infection
MDR-TB Multiple drug-resistant TB
MGIT Mycobacteria growth indicator tube
MICs Minimum inhibitory concentrations
NAAT Nucleic acid amplification technology
PAS Para-amino salicylic acid
SAT Self-administered therapy modified
TB Tuberculosis
XDR Extensive drug resistant
20.1 Introduction
TB can involve all organs except the nails, hair and teeth [Ekaterina Kulchavenya.
Ther Adv Infect Dis. 2014 Apr;2(2): 61–70]. Pulmonary TB is the most common
form of TB. The symptomatology of TB has to do with its pathogenesis, the specific
organ system involved and the duration of the disease.
Primary TB, which occurs in the Mycobacterium-naive individual, usually
presents with a frequently occult primary focus in the peripheral lung parenchyma
(Stead’s focus) accompanied by a pleural effusion; or by a primary focus—fre-
quently inapparent—in the lower part of the upper lobe or the upper part of the lower
lobe (Ghon’s focus) accompanied by lymphadenopathy. Other manifestations of
20 Clinical Aspects and Principles of Management of Tuberculosis 357
primary TB such as erythema nodosum or phlyctenular conjunctivitis may be

present. In young children with immature cellular immunity, or in immunocompro-
mised individuals, the process may evolve to progressive primary disease.
Symptoms and signs relate to the compressive effect of enlarged lymph nodes on
adjacent structures (collapse or hyperinflation of a lobe or segment), or rupture of
caseating lymph nodes into a bronchus, with bronchiectasis later supervening in such
a lobe.
Post-primary disease, the typical adult form of TB, usually occurs by reactivation
of a dormant focus–or less frequently, by reinfection; the better aerated lung apices
are preferentially involved. The classical triad of the constitutional symptoms of TB
(fever, night sweats, weight loss)—most usually of several weeks’ duration—is
present in a high proportion of patients (Heemskerk et al. 2015).
The extent of disease at the time of first diagnosis may be variable, from patchy
consolidation to a large cavity. This not only reflects the protean pulmonary forms of
the process but also has to do with host immunity, the virulence of the strain and,
most of all, with the time elapsed between disease onset and diagnosis.
Cavitation is an important milestone in the timeline of the post-primary pulmo-
nary tuberculosis. With ingress of O2-rich air into a fresh cavity (caused by the
coughing out of a caseous collection), the aerobic Mycobacterium is presented with
an opportunity to multiply manifold in an aerobic environment: the infectiousness of
the subject (and the possibility of a positive diagnosis on sputum testing) commen-
surately increases (Golub et al. 2006). Cavitation also increases the propensity to
haemoptysis (which may sometimes be massive) and that of cavitary colonization
with a fungal ball (mycetoma) later on in healed disease. One third of all individuals
affected with post-primary TB die within a few months (“galloping consumption”)
(Reported tuberculosis in the United States, 2013 2014). The remainder either go on
to develop a chronic pattern with a more gradual progressive decline or undergo
spontaneous remission of the process and restoration to health. With chronicity,
fibrosis is common.
Disseminated TB, which often casts “miliary” shadows in the lungs, occurs due to
haematogenous dissemination following either primary or post-primary TB. The
name derives from the resemblance of the widespread granulomatous lesions to
millet seeds (seen in pathological specimens as 1–2 mm yellow granules).
Symptoms may be nonspecific and depend on the site and degree of organ involve-
ment. Hepatosplenomegaly, lymphadenopathy and pleural effusions may be present.
Fundus examination, often neglected, will frequently show choroid tubercles, which
are virtually diagnostic of the condition in a likely setting.
Diagnosis often hinges on microbiology from broncho-alveolar lavage (BAL) or
on biopsy of involved organs (Lewinsohn et al. 2017) though it may occasionally be
made on sputum (sponaneously expectorated or induced).
Disseminated TB Disseminated TB should always raise suspicion for an

accompanying immune deficiency such as HIV disease. Sometimes an occult
disseminated form of TB (“cryptic miliary TB”) presents as a “fever of unknown
origin”, usually in the elderly or immunocompromised patients. In difficult cases
358 A. Hasan et al.
(as with the classic miliary form), bone marrow or liver biopsy with Mycobacterial
cultures may be diagnostic.
Unlike the post-primary form of disease, untreated miliary TB is predictably fatal.
Extra-Pulmonary Tuberculosis
Extra-pulmonary tuberculosis has been increasingly seen since the advent of the HIV
epidemic. TB can involve any organ of the body (other than nails, hair and teeth).
Organ systems typically involved include pleura (pleural effusions), lymph nodes
(generally cervical or mediastinal), airway (endobronchial tuberculosis), skeletal
(joint or vertebral involvement, sometimes with a psoas abscess), gastrointestinal,
genitourinary, meningeal and pericardial involvement. Symptoms pertain to the
system involved, but diagnosis may be difficult unless a high degree of suspicion
is maintained. Tissue or fluid samples from the involved organs with staining,
Mycobacterial cultures or molecular probe assays are often diagnostic although
radiological patterns in specific cases may be very suggestive of the process
(e.g. vertebral involvement with a paraspinal abscess) (Lewinsohn et al. 2017).
Diagnosis
The diagnosis of tuberculosis, especially in the developed nations, rests on the
keystone of suspicion. Physical examination is generally unrewarding. Auscultation
of the lung typically underestimates the lung problem relative to that seen on
imaging. With a pleural effusion, breath entry is typically decreased, with a flat
note over the effusion. Cavernous bronchial breathing may be detected over a cavity,
and tubular bronchial breathing over an area of consolidation or dense fibrosis.
The radiologic features in the various forms of pulmonary TB have already been
discussed above under the relevant heads.
Microbiological isolation of the Mycobacterium tuberculosis on culture has been
the only definitive test for TB thus far, though it could be argued that molecular
probes for specific mycobacterial genes and other technologies (discussed below and
also elsewhere in this book) offer viable and more attractive options for diagnosis.
Presumptive diagnosis is typically achieved by microscopy (usually of sputum, but
also of fluid obtained by BAL and other means) (Pai et al. 2016).
Mycobacteria, not being stainable by “conventional” stains, require a modified
acid stain (the Ziehl-Neelsen method) in order to be visualized; LED microscopy
though relatively expensive, by making the bacilli easy to see under low power,
enables more fields to be scanned in a shorter period of time, and thus increases the
sensitivity of microscopy several-fold (Bhalla et al. 2013).
Conventional culture methods are slow (4–6 weeks, less by radiometric assays),
and frequently there is a compelling need to start appropriate anti-tubercular therapy
as early as possible. Rapid culture techniques are now in universal use. They use
liquid rather than solid media. The Mycobacteria growth indicator tube (MGIT)
method takes days rather than weeks and facilitates drug sensitivity testing.
Since relapses (see below) occur in approximately 5% of patients after 6 months

of standard therapy, it is a moot point whether establishment of minimum inhibitory
concentrations (MICs) in the presence of a “critical concentration” of drug is
relevant in selected cases (Colangeli et al. 2018).
Automated nucleic acid amplification technology (NAAT) has the potential of
amplifying even a fragment of mycobacterial DNA rapidly (in 2 h) and has
revolutionized TB diagnosis. In its currently available form, the GeneXpert test is
intended specifically to diagnose Mycobacterium tuberculosis (and thereby differ-
entiate it from other Mycobacteria such as environmental Mycobacteria), and also
presumptively diagnose rifampicin resistance.
Though not invariable, rifampicin resistance also implies INH resistance.
Together, rifampicin and INH resistance fulfil the diagnosis of multiple drug-
resistant TB (MDR-TB). Thus a positive GeneXpert test makes a presumptive
diagnosis of MDR-TB (Steingart et al. 2009). The test is more sensitive than direct
microscopy and extremely specific (Steingart et al. 2014). The test is not infallible,
however, and any result that is discordant with the clinical picture should either be
repeated or further information sought using another modality. False-positive NAAT
results can occur due to laboratory contamination. Importantly, NAAT can detect
nucleic acid from dead non-viable bacilli, and so a positive test after completion of
the course of treatment has no relevance in that situation (Boyles et al. 2014) nor is it
of relevance in monitoring response to treatment (Friedrich et al. 2013). It is
important to realize that NAAT is not a substitute for AFB smear and culture testing;
culture is essential for species identification and for drug susceptibility testing
(Cheng et al. 2005). A more sensitive version of Xpert has been developed.
The line probe assays (LPA) can permit rapid identification of specific gene
markers associated with rifampicin resistance alone or in combination with isonia-
zid, and provide clinically relevant information about the level of INH resistance
(low level associated with the INH-A gene; versus high level associated with the
kat-G gene) (WHO treatment guidance for drug resistant tuberculosis 2016).
Definitive diagnosis has traditionally hinged on the culture characteristics of
MTB: species discrimination can be achieved on the basis of colony morphology
as well as on the basis of biochemical tests performed on the culture. Conventional
culture (Lowenstein-Jensen medium and its variants) is capable of detecting as few
as ten bacteria per millilitre with the sensitivity and specificity of sputum culture
approximating 80 and 98%, respectively (Morgan et al. 1983). Following initial
culture, species identification is done by any of the following techniques: bio-
chemical methods (testing, nucleic acid hybridization probes, high-pressure liquid
chromatography or mass spectrophotometry). Drug sensitivity testing is subse-
quently carried out, usually at a reference laboratory (Shinnick and Good 1995) to
first-line anti-tubercular agents, but sometimes to second-line agents as well when
resistance to components of first-line anti-tubercular agents has been documented.
In individuals living with HIV who have CD4 counts <100 cells/mm3, or those
who are immunocompromised, Mycobacterial cultures of blood and urine should
complement conventional testing. Point-of-care urinary detection of
lipoarabinomannan, a component of the Mycobacterial cell wall (urine LAM
360 A. Hasan et al.
test) may be useful for HIV-infected individuals, and its use may possibly confer a
mortality benefit (Reported tuberculosis in the United States, 2013 2014). The
future of point-of-care devices is next-generation sequencing, and, perhaps,
innovations like detection of volatile organic compounds in exhaled breath
(Garcia-Basteiro et al. 2018)
Serological tests (tests for antibodies against TB) have poor sensitivity and
specificity; false-positive tests are ubiquitous, and serologic testing for the diagnosis
of TB is discouraged by the WHO.
A pleural fluid adenosine deaminase (ADA) >35 U/L has been reported to be 93%
sensitive and 90% specific for the diagnosis of pleural TB in lymphocytic exudates
(Garcia-Zamalloa and Taboada-Gomez 2012).
20.2 TB in Special Circumstances
20.2.1 TB in Childhood
TB infection can rapidly progress to disease in infants and young children (progres-
sive primary TB). Mortality is high in early childhood because of the severe forms of
TB (such as miliary TB and meningeal TB) that occur more frequently in young
children. Progressive primary TB is a serious consequence of primary tuberculosis.
Consolidation or bronchopneumonia is more common than cavitary disease in
children. Haematogenous dissemination leading to miliary TB is also more common
in infants and young children. Tubercular pleural effusion, however, is less common
in children below 5 years of age.
Central nervous TB is the most serious form of childhood TB (Prevention 2014).
The presentation is usually with nonspecific symptoms such as fever, headache,
drowsiness and irritability, but in advanced cases, vomiting, neck rigidity, seizures,
focal neurologic deficit and coma may supervene.
Since sputum samples for diagnosis are difficult to obtain in young children
(children tend to swallow sputum rather than expectorate), gastric washings can
rather be sent for microbiological examination. A PPD test that is greater than 10 mm
in BCG-unvaccinated children and > 15 mm in BCG-vaccinated children is consid-
ered positive. (Targeted tuberculin testing and treatment of latent tuberculosis
infection. This official statement of the American Thoracic Society was adopted
by the ATS Board of Directors, July 1999. This is a Joint Statement of the American
Thoracic Society (ATS) and the Centers for Disease Control and Prevention (CDC).
This statement was endorsed by the Council of the Infectious Diseases Society of
America. (IDSA), September 1999, and the sections of this statement 2000.)
The principles of treatment of tuberculosis in children are similar to that of adults.
Paediatric TB patients should be monitored carefully for ethambutol-induced ocular
toxicity, owing to the fact that children are less likely to report visual impairment
than adults.
In a prospective study of children on multidrug-resistant tuberculosis, chil-
dren had lower serum concentrations in spite of higher dosing of moxifloxacin, a
fact that was ascribed to increased drug elimination in children. Quinolones have
also been known to rarely cause arthropathy and tendon rupture. More recent studies
have shown that most quinolones are quite well tolerated but very specific and
narrow indications are present for quinolone use in children (Patel and Goldman
2016).
20.2.2 TB in Pregnancy
Tuberculosis in pregnancy has important implications for the mother and child (Loto
and Awowole 2012).
The diagnosis of tuberculosis in pregnancy can be challenging, since many of the
symptoms of early TB may be confounded by pregnancy; for instance, the normal
weight gain in pregnancy may temporarily mask the associated weight loss. Chest
radiograph is not an absolute contraindication and when necessary may be carried
out with “abdominal shielding” of the fetus.
The complications of TB in pregnancy include early spontaneous abortion,
intrauterine growth retardation, preterm delivery, low birth weight and increased
neonatal mortality. Congenital TB is rare, but can be associated with high perinatal
mortality.
First-line oral drugs, i.e. isoniazid, rifampicin, ethambutol and pyrazinamide, can
all be used during pregnancy safely. Streptomycin and other aminoglycosides have
been proven to be potentially teratogenic in pregnancy. They are also capable of
causing eighth nerve paralysis, with deficits ranging from mild hearing loss to
bilateral deafness (Loto and Awowole 2012). Concerns have been raised about the
use of fluoroquinolones in pregnancy even though the weight of the evidence seems
to point toward safety (Acar et al. 2019)
20.2.3 TB in HIV
TB is considered an AIDS-defining disease [Centers for Disease Control and

Prevention. Appendix A: AIDS-Defining Conditions. MMWR. 2008;57(RR10):9].
The risk of developing TB is estimated to be between 16 and 27 times greater in
people living with HIV than among those without HIV infection. HIV and TB have a
salutary impact upon each other and expedite each other’s progression. The risk of
progressing from latent to active TB is estimated to be between 12 and 20 times
greater in people living with HIV than among those without HIV infection (Global
TB report 2017).
Against the backdrop of HIV infection, the rate of progression of TB from the
acquisition of infection to full-blown disease may take weeks, rather than years—as
is the case in HIV-negative individuals (Mayer and Dukes Hamilton 2010). It is
important to realize that TB can occur at all “stages” of HIV disease. The clinical
presentation of HIV patients who have relatively high CD4 counts is no different to
that which occurs in individuals without HIV. Such individuals manifest with the
362 A. Hasan et al.
classical manifestations of post-primary tuberculosis such as upper lobe infiltrates.

On the other hand, lower lobe involvement, pleural effusions and intrathoracic
lymphadenopathy are common in individuals with low CD4 counts (typically
<200/μL). Indeed the chest X-ray may sometimes only show vague interstitial
infiltrates or even be normal (Mayer and Dukes Hamilton 2010). Taken together,
the fact that in advanced HIV disease (a) the sputum is less likely to be positive for
AFB (b) the PPD is usually non-reactive (c) the X-ray abnormalities are likely to
represent other opportunistic infections or even non-infective conditions associated
with HIV disease and (d) histopathology of involved tissues is characterized by lack
of typical granulomata; these considerations make the diagnosis extremely challeng-
ing in this setting. In addition, there is increased frequency of extra-pulmonary TB
among HIV-positive individuals.
Frequently, a positive NAAT (GeneXpert) test is the justification for starting
treatment. However it must be cautioned that even a negative NAAT test cannot
convincingly rule out disease, and, given that delays in treatment may be fatal, the
decision to initiate treatment is sometimes taken on clinical grounds.
20.3 Treatment of TB
The treatment of tuberculosis differs from the treatment of most other infections
in certain respects. Multiple drugs are required in a regimen since spontaneous
mutations can lead to resistance to the action of one or more drugs. Also, because
the bacterium is relatively slow growing, it takes longer to achieve bacteriologic
sterility. Multidrug therapy, the norm for TB, has been very effective, with a
modest rate of relapse (Colangeli et al. 2018).
The standard four-drug regime (“short course”) comprises a 2-month intensive
phase with the four front-line drugs (rifampicin, isoniazid, ethambutol and
pyrazinamide), followed by a continuation phase with the drugs (minus
pyrazinamide and possibly ethambutol) for a further 4 months.
The four front-line drugs have been chosen based on their efficacy in rapidly
reducing the number of organisms initially (during the initial phase), thus reducing
infectivity; and on their sterilizing potential (the ability to eradicate viable bacteria
completely and so prevent relapses); and their relative lack of side effects (see
Table 20.1). These drugs are combined into regimens.
The six classes of second-line agents (fluoroquinolones, the aminoglycosides,
capreomycin, ethionamide and prothionamide, para-amino salicylic acid (PAS),
cycloserine and terizidone) have a rather lower efficacy and a higher propensity
for side effect and drug intolerance. Several other antibiotics of uncertain efficacy
have also been categorized as anti-tubercular agents and have a role in treating
multidrug-resistant TB (MDR-TB) (see Table 20.2).
Two new agents, bedaquiline (a diaryl-quinolone) and delamanid
(a nitroimidazole), are now approved for the treatment of MDR-TB.
The WHO recommends using a daily regimen and fixed-dose combinations
(Guidelines for treatment of drug-susceptible tuberculosis and patient care 2017).
Table 20.1 First-line agents for standard first-line anti-tubercular therapy

Adult
Drug daily dose Comment
Isoniazid 5 mg/kg Give pyridoxine 10 mg daily to prevent peripheral neuropathy; if
established peripheral neuropathy, give pyridoxine 50–75 mg
daily
Protect tablets from light
Rifampicin 10 mg/kg Preferably given 30 min before food
Pyrazinamide 25 mg/kg Avoid with active hepatic disease (see further discussion on drug-
induced hepatitis, below); adjust dose for renal function
Ethambutol 15 mg/kg Adjust dose for renal function
Ocular examination to be done before and periodically during
treatment
Administration of the drugs on a daily basis both during the intensive and during the
continuation phase is preferable although daily administration during the initial
phase and intermittent (thrice weekly) administration during the continuation
phase is also acceptable (Guidelines for treatment of drug-susceptible tuberculosis
and patient care 2017). Intermittent administration of drugs (especially in the initial
phase) is not offered in the presence of HIV disease.
If at the end of the intensive phase the bacteria are sensitive to the three most
important agents (isoniazid, rifampicin and pyrazinamide), ethambutol may be
discontinued (Nahid et al. 2016). However, drug testing for three of the four front-
line drugs for each patient at the end of the intensive phase is impractical, and limited
testing (GeneXpert) is usually performed initially in those patients whose sputum
remains positive. There is evidence to show that extending the continuation phase by
7 months (making up a total duration of treatment of 9 months) helps in preventing
relapse in those patients who have cavitation on initial chest X-rays; and also in those
who show positive cultures at the end of the initial phase of treatment (Benator et al.
2002); as well as in those whose medication given during the initial phase did not
include pyrazinamide.
Anti-tubercular drugs require to be supplemented with carefully monitored ste-
roid therapy in two circumstances: tubercular meningitis (a short course of dexa-
methasone or prednisolone is typically given, tapered over 6 to 8 weeks) and
tuberculous pericarditis (Guidelines for treatment of drug-susceptible tuberculosis
and patient care 2017).
Since many patients cannot be relied upon to regularly take their drugs, and
irregular intake of drugs has the potential to result in drug resistance (see Treatment
Failure and Relapse; and Drug Resistant TB, below), it is imperative to ensure
adherence to the regimen. Directly observed therapy, short-course (DOTS) is the
direct observation by another person, of the patient taking the medication
(Chaudhuri 2017). The use of DOTS has improved adherence in subgroups of
patients such as TB with HIV or if DOTS was at the site of DOTS centre or provider
location.
364 A. Hasan et al.
Table 20.2 First and second-line anti-tubercular agents and their adverse drug reactions (ADRs)
Drugs Common ADRs Uncommon ADRs Rare ADRs
Isoniazid Asymptomatic Hepatitis, cutaneous Fever, giddiness,
elevation of serum hypersensitivity, convulsions, optic
hepatic enzymes peripheral neuropathy neuritis, mental
symptoms, haemolytic
anaemia, aplastic
anaemia, lupoid reactions,
arthralgia, gynaecomastia
Rifampicin Pruritus Hepatitis, cutaneous Shortness of breath,
hypersensitivity, shock, haemolytic
gastrointestinal anaemia, acute renal
reactions, failure, thrombotic
thrombocytopenia, thrombocytopenic
febrile reaction, “flu purpura
syndrome”
Pyrazinamide Anorexia, nausea, Hepatitis, vomiting, Sideroblastic anaemia,
flushing, arthralgia, cutaneous gout
photosensitization reactions
Ethambutol Retrobulbar neuritis, Peripheral neuropathy
cutaneous reactions,
arthralgia
Streptomycin Cutaneous Vertigo, ataxia, Clinical renal failure,
hypersensitivity, deafness aplastic anaemia
giddiness,
numbness, tinnitus
Thioacetazone Gastrointestinal Hepatitis, erythema Agranulocytosis
reactions, cutaneous multiforme, exfoliative
hypersensitivity, dermatitis, haemolytic
vertigo, anaemia
conjunctivitis
Amikacin/ Hearing damage, Clinical renal failure
kanamycin/ vestibular
capreomycin disturbance,
deranged renal
function tests
Ofloxacin/ Gastrointestinal Anxiety, dizziness, Convulsion, cutaneous
ciprofloxacin reactions, insomnia headache, paresthesia, hypersensitivity
tremor
Ethionamide/ Gastrointestinal Hepatitis, peripheral Convulsion, depression,
prothionamide reactions neuropathy alopecia, hypothyroidism,
impotence,
gynaecomastia
Cycloserine Dizziness, Psychosis, convulsion Sideroblastic anaemia
headache,
depression, memory
loss
Para- Gastrointestinal Hepatitis, drug fever Hypothyroidism,
aminosalicylic reactions haematological reactions,
acid generalized
hypersensitivity,
malabsorption

First line anti-tubercular agents
Self-administered therapy (SAT) modified with some form of monitoring

incorporated into the process shows some promise. Although DOTS could be
administered by a family member, the preferred person is a trained lay provider or
healthcare worker (Guidelines for treatment of drug-susceptible tuberculosis and
patient care 2017).
The treatment of TB in HIV disease merits special consideration. Antiretroviral
therapy (ART) should be started irrespective of the clinical stage of HIV disease and
CD4 count. Anti-tubercular therapy (ATT) should precede the commencement of
ART. ART should then be commenced as soon as it is evident that ATT is being
tolerated (typically between 2 weeks and 2 months).
The immune reconstitution inflammatory syndrome (IRIS) is sometimes seen with
the returning immune competence that follows ART; an increased immune response
to tubercle bacilli or antigens occurs. IRIS usually occurs between 1 and 3 months of
the commencement of ART and is associated with a decrease in viral load and an
increase in CD4+ T cell count. There is a worsening in the clinical picture weeks or
months into treatment, with an increase in the size of lymph nodes, development of
pleural effusions and features of noncommunicating hydrocephalus in a patient with
TB meningitis.
Although most patients with IRIS have mild to moderate symptoms, IRIS,
especially when related to TB meningitis, can be life-threatening. An “unmasking
IRIS” may occur in patients with unsuspected TB (Raviglione 2017).
Treatment of IRIS includes addition of steroids (which probably work by decreas-
ing the levels of pro-inflammatory cytokines) and the continuation of ATT and ART.
With both ATT and ART on board, drug-drug interactions especially involving
rifampicin (by induction of the cytochrome P450 system) may occur. Rifamycins
induce the metabolism of non-nucleoside reverse transcriptase inhibitors and prote-
ase inhibitors. Rifampicin is a more potent inducer of the cytochrome P450 system
than rifapentine, which in turn is more potent than rifabutin. Despite potential drug
interactions, rifamycins should nevertheless be included in TB regimens, with
dosage adjustment if necessary. Rifabutin is the preferred rifamycin in
HIV-positive individuals.
20.4 Alternative Treatment Approaches
Host-directed therapy to modulate the immune response to TB is an active area of

research (Kaufmann et al. 2014). The use of an anti-diabetic drug—metformin, for
example—is being investigated. Also, studies of autologous mesenchymal stromal
cell infusions is being investigated in MDR and XDR-TB (Skrahin et al. 2014).
An approach using high-altitude sanatoria was also explored (Murray 2014) in a
throwback to the pre-anti-tubercular chemotherapy era. Study using atypical
Mycobacterial injection in tuberculous did not show any major outcome differences
from placebo although steroids did decrease the subsequent development of
constrictive pericarditis (Mayosi et al. 2014).
366 A. Hasan et al.
Given the role that vitamin D plays at the level of the antigen-presenting cell, the
role of vitamin D deficiency as a possible player in TB predisposition has recently
come into focus; indeed a recent meta-analysis has revealed that vitamin D supple-
mentation in fact did not affect time to sputum culture conversion overall, though it
did accelerate sputum culture conversion in patients with multidrug-resistant pulmo-
nary tuberculosis (Jolliffe et al. 2019).
20.5 Critical Care of Patients with TB
Patients with TB may be admitted to an ICU for a variety of reasons, including

respiratory failure, multiorgan failure, decreased consciousness associated with
tubercular meningitis, pneumothorax, cardiogenic shock (due to massive pericardial
effusion) and liver or kidney failure due to a drug reaction induced by ATT.
Confluent tuberculous bronchopneumonia and TB ARDS are less common causes
of respiratory failure (Hagan and Nathani 2013).
Tuberculosis treatment is especially challenging in critically ill patients due to
potential for poor gastric absorption and the high rates of organ dysfunction and
drug toxicity in this subset of patients. Deciding the optimal regimen and dosing
becomes challenging in the presence of concomitant hepatic and renal failure.
In a recent study done on TB patients requiring ICU admission, mortality was
72.5% (Tatar et al. 2018).
20.6 Treatment Failure and Relapse
Treatment failure is suspected when the sputum remains positive for AFB at the end
of the first 3 months of treatment. It occurs due to factors that may involve incorrect
regimens or dosing or indeed due to malabsorption or poor-quality drugs
(Lambregts-van Weezenbeek and Veen 1995). Patients are regarded as treatment
failures when a microbiologic indicator like sputum smear/culture for TB remains
positive after 3–4 months of starting treatment or comes back positive after a period
of remaining negative (fall and rise phenomenon) (Prasad et al. 2018). Patients may
respond for a short time after starting treatment or do not respond to treatment from
the inception. In any case the implication of treatment failure is drug-resistant TB
(see below), and strenuous efforts must be made to look for microbiological resis-
tance to first—and, in the proper context, to second-line agents—by the available
tools. Relapse, on the other hand, is the recurrence of TB after completion of
treatment that has been successful, with documented culture negativity. Relapse
may be due either to a reinfection or reactivation of the dormant bacillus. Drug-
resistant TB is to be considered in these settings. The implications of relapse are not
as grave, the isolates generally being drug sensitive.
In either case, the exact regimen chosen should be based upon the drug sensitivity
pattern. Specific recommendations upon the construction of such a regimen are
available at WHO treatment guidelines for drug-resistant tuberculosis 2016 update

(http://www.who.int).
20.7 Drug-Resistant TB
Drug-resistant TB is a major global problem and is a threat to the success of the End
TB strategy. MDR-TB or multidrug-resistant TB (MDR-TB) is defined by resistance
of M. tuberculosis against to at least rifampicin and isoniazid. Since these two drugs
are the most effective drugs available against TB, resistance to both considerably
shortens the odds of complete cure.
XDR-TB or extensively drug-resistant TB is defined as resistance to rifampicin
and INH plus resistance to at least one fluoroquinolone and one second-line
injectable agent (amikacin, kanamycin or capreomycin).
Some of the reasons that lead to drug resistance have been mentioned in the
section above. However the most important of these are failure to comply with the
treatment regimen on the part of the patient and the addition of a single drug
(“monotherapy”) to a failing regimen; or a situation of unrecognized primary (that
which is present at the start of therapy) or secondary (that which develops during
treatment) drug resistance.
INH mono-resistance of itself probably does not impact the outcome of therapy
provided that ethambutol and possibly pyrazinamide are used for the entire duration
of therapy. Consideration should be given in this case to extending the duration of
treatment to 9 months from the standard 6 (Sadanand 2011). For reasons already
discussed, rifampicin-resistant individuals should be managed as MDR-TB patients.
In such patients it is not unusual to encounter resistance to additional drugs
(e.g. ethambutol).
It has been shown that clinical cure is possible in patients with MDR and even
XDR-TB (WHO drug resistant TB 2016). However, owing to a variety of reasons
not the least of which is drug intolerance, only about half of MDR-TB and a third of
XDR-TB patients ever complete therapy. The WHO estimates there are over
600,000 MDR-TB or rifampicin-resistant cases annually (Global TB report, WHO
update 2017; wwww.who.int). The treatment of MDR-TB and XDR-TB is complex
and requires specialized knowledge and close monitoring to ensure rational and safe
therapy (Lange et al. 2018).
The choice of drugs for treatment of MDR-TB is frequently difficult. Formulation
of drugs into regimens is facilitated by the organization of drugs into groups:
Group A ¼ levofloxacin/moxifloxacin, bedaquiline, linezolid.

Group B ¼ clofazimine, cycloserine/terizidone.
Group C ¼ ethambutol, delamanid, pyrazinamide, imipenem-cilastatin, meropenem,
amikacin (streptomycin), ethionamide/prothionamide, p-aminosalicylic acid.
The WHO makes specific recommendations for short and long MDR-TB
regimens. In the WHO’s 2018 update (WHO treatment guidelines for multidrug-
368 A. Hasan et al.
and rifampicin-resistant tuberculosis 2018), for those MDR-TB patients “who have
not been previously treated for more than one month with second-line medicines
used in the shorter MDR regimen or in whom resistance to fluoroquinolones and
second-line injectable agents has been excluded,” the WHO permits a shorter
MDR-TB regimen of 9–12 months.
For MDR/RR (RR¼ rifampicin resistant) TB patients on longer regimens, the
WHO recommends that “all three Group-A agents and at least one Group-B agent
(should) be included to ensure that treatment starts with at least 4 TB agents likely to
be effective, and that at least three agents (are) included for the rest of treatment after
bedaquiline is stopped. If only one or two Group A agents are used, both Group B
agents are to be included. If the regimen cannot be composed with agents from
Groups A and B alone, Group C agents are added to complete it. Kanamycin and
capreomycin are not to be included in the treatment of MDR/RR-TB patients on
longer regimens” (WHO treatment guidelines for multidrug- and rifampicin-resistant
tuberculosis 2018). A detailed discussion about the treatment of MDR-TB is avail-
able at the foregoing resource.
Novel anti-tubercular agents (e.g. bedaquiline and delamanid) have been
discussed elsewhere in this text.
20.8 The Sequelae of Tuberculosis
The remodelling of the lung that follows TB can significantly contribute to morbidity
and mortality. Haemoptysis is frequently one of the remote sequelae and can on
occasion be life-threatening. An unresolved cavity is frequently the cause, and
bleeding from an unsupported blood vessel in the cavity wall or in a post tubercular
bronchiectatic segment can be a source of troublesome bleed. Cavities colonized by
fungi (aspergillomas), broncholiths eroding into the bronchial wall and rarely
expansion and subsequent rupture of an unsupported blood vessel in the cavity
wall (Rasmussen’s aneurysm) can cause massive haemoptysis (van den Heuvel
and van Rensburg 2006). A scar carcinoma sometimes develops in an area of fibrosis
and may sometimes be difficult to distinguish from a dense fibrotic infiltrate (Gao
et al. 2015). Post-tubercular bronchiectasis is predisposed to when caseation necrosis
and inflammation with retention of secretions leads to destruction of bronchial walls
with ensuing permanent dilatation. Sometimes compression of bronchial lumen by
enlarged lymph node can produce bronchiectasis by a different mechanism. Post-
primary TB involves the upper lobes of the lung, and so the gravitationally
advantaged upper lobes do not usually undergo a process of chronic and recurrent
infection unlike the lower lobes that are involved by bronchiectasis of other
aetiologies.
Mycetoma (fungal ball) is a mass of fungal hyphal material that grows within a
cavity. Aspergillus fumigatus is the most common aetiological agent, but other fungi
such as Mucor or Fusarium can also cause a fungal ball to develop (Rippon 1988).
Such a mycetoma is usually asymptomatic but can sometimes lead to haemoptysis.
No treatment required in asymptomatic cases, but surgical treatment needs to be

considered in patients with massive or recurrent haemoptysis.
20.9 Side Effects of Treatment
A list of adverse effects associated with anti-tubercular agents appears in Table 20.2.
Side effects are possible with any of the anti-tubercular agents, and on rare
occasions, they may be serious and even fatal. The risk of liver injury with anti-
TB therapy is always a concern. Among the four first-line agents INH, rifampicin
and pyrazinamide are all capable of causing hepatotoxicity. Isoniazid is capable of
causing asymptomatic mild elevation in transaminases in 15–20% as well as fatal
severe acute hepatitis in 0.05–1%. Jaundice with liver injury can occur in 0.5–1%.
Peripheral neuropathy due to pyridoxine deficiency can occur with INH therapy in
up to 6.5% of elderly individuals (Ruan et al. 2018); pyridoxine supplementation is
required for all patients taking INH (John 2019). Factors increasing the chances of
INH hepatotoxicity include age, female gender, alcohol use, prior hepatitis, concur-
rent use of cytochrome-inducing agents and slow acetylator status (Sharma et al.
2016). Acute INH toxicity can present as seizures due to decreased GABA levels.
INH is metabolized to several metabolites including the hepatotoxic metabolite
hydrazine, toxic-free radicals and mono-methylhydrazine. Degradation by acetyla-
tion occurs via N-acetyltransferase-2 gene (NAT2); a deficiency of the NAT enzyme
can lead to accumulation of hepatotoxic metabolite. Ten percent of patients with
anti-tubercular drug-induced hepatotoxicity show progression to acute liver failure;
the rest are self-limited (Badrinath and John 2018).
If the serum bilirubin rises to 3 mg/dL or serum transaminases exceed more
than five times the upper limit of normal (or exceed three times the upper limit of
normal in a symptomatic patient), all drugs should be stopped (Nahid et al. 2016).
The decision on what regimen to use on restarting should be individualized. The
regimen, even if retailed, might then still include one or more potentially hepatotoxic
drugs, and these should be restarted one at a time, only when liver function tests
return to their baseline (e.g. the transaminases fall to less than twice normal). In
general, a cholestatic type of derangement would prompt addition of INH first; with
a non-cholestatic picture, rifampicin could first be added (Sterling). Needless to say,
careful monitoring with frequent testing of serum liver function is required. Most
physicians would omit pyrazinamide when restarting ATT except in the mildest of
cases or with other compelling reasons (Sterling).
Patients receiving ethambutol (EMB) should be questioned regarding their vision
at each monthly visit. Monthly visual acuity and colour vision discrimination testing
is recommended in those patients in whom EMB doses of greater than 15–20 mg/kg
are being used, those who have been taking ethambutol for longer than 2 months and
those with renal insufficiency in whom the levels of drug may be expected to rise
(Blumberg et al. 2003).
370 A. Hasan et al.
For patients who require a regimen with no hepatotoxic agents, potential agents
include ethambutol, levofloxacin or moxifloxacin, an injectable agent and other
second-line oral drugs. The optimal choice of agents and duration of treatment
(at least 18–24 months) is uncertain. (See “Antituberculous drugs: An overview”,
section on “Second-line agents”.)
20.10 The Prevention of TB
In the end, efforts to finally eradicate TB will need to hinge upon preventative
aspects as much as upon its treatment. These strategies would include currently
established practices, as well as novel strategies. The former include the following.
20.10.1 Vaccines
Tuberculosis is now the primary cause of death from infectious diseases exceeding
HIV, AIDS and malaria. The WHO estimates 10.4 million new cases and 1.7 million
deaths every year (WHO Global TB report 2017). Of these, 0.4 million are HIV
patients who die mostly in LMIC (low- and middle-income countries) (WHO Global
TB report 2017; www.who.int). Within the next 16 years, the goal is to reduce TB
incidence by 90% but the progress has been painfully slow. The most effective way
to reduce the burden of TB in the community, to date, has been to identify patients
affected with TB as early as possible and to treat them effectively with anti-
tubercular therapy.
The role of vaccines in this regard, though potentially vital, has been disappoint-
ing. Of all vaccine tested to date, the bacillus Calmette-Guerin (BCG)—a live
attenuated vaccine that was made in 1919 after an incredible 11 years and
231 sub-cultures (Hasan 2014)—is still the most effective vaccine we have today.
Yet its effect has been inconsistent, ranging from 80% to zero protection; several
factors including geographical differences appear to play a role (British adolescents
had better responses than South Indian participants perhaps due to background
exposure to MTB or atypical mycobacteria). The efficacy of BCG has been most
manifested in its protection of infants and young children from the dangerous forms
of TB (such as disseminated “miliary” TB and TB meningitis). Also, its effects
appear to wane with time (Hasan 2014).
The BCG is not a single vaccine. Several strains—all derived from the original
1921 BGC strain—are in use, which also partly explains its variable efficacy.
In 1998, the Sanger Centre in Cambridge, Britain and Paris’s Pasteur Institute in
France cracked the genetic code for the old H37Rv strain of tubercle bacillus, and
then, the genome of highly virulent CDC1551 (the “Oshkosh” strain) was mapped at
the TIGR (Fine 1989). These developments have led to the possibility of exciting
new candidate vaccines, several of which are in fact in the pipeline.
20.10.2 Treatment of Latent TB Infection (LTBI)
It has been estimated that one-third of the world has been infected with TB bacteria.
A relatively small number of immunocompetent individuals affected with LTBI will
ever progress to active disease. Reactivation of TB occurs when the immune system
is compromised by external factors such as steroids or other immunosuppressants or
by immune deficiency syndromes, which enables the latent (dormant) bacteria to
“awaken” causing active disease. There is unfortunately no test that can currently
predict which of these individuals will progress to active disease immunocompro-
mised individuals have a high possibility of doing so.
Given that perspective, treatment for latent TB infection is most relevant for
children under 5 years, the elderly and individuals with HIV infection. To eliminate
TB from the planet, a multipronged strategy incorporating early and effective
treatment, vaccination and elimination of dormant reservoir of latent TB will be
essential.
LTBI treatment with INH (“chemoprophylaxis”) is still the mainstay for
treating latent TB infection but given that treatment extends to 6 months, and
that there is one death from hepatotoxicity for every 25,000–40,000 cases with
such treatment, alternate strategies including rifamycin-based regimens are being
explored (Rangaka et al. 2015).
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Tuberculosis Vaccine: Past Experiences
and Future Prospects 21
Gurpreet Kaur, Deepjyoti K. Das, Sanpreet Singh, Junaid Khan,
Mohammad Sajid, Hilal Bashir, Mohammad Aqdas, Shikha Negi,
Uthaman Gowthaman, and Javed N. Agrewala
Abstract
Vaccines are the best prophylactic measure that have eradicated several diseases
like polio, measles, and small pox. Currently, efficient vaccines are unavailable
for many dreaded diseases, including tuberculosis (TB). TB is caused by an
intracellular pathogen Mycobacterium tuberculosis (Mtb) and is responsible for
about two million deaths and nine million new cases annually. The WHO has
announced TB as a global emergency in the wake of emerging multidrug-
resistant, extremely drug-resistant, and totally drug-resistant strains of Mtb. An
alarming increase in the number of TB cases around the world and its
co-occurrence with HIV has further complicated the problem. Additionally,
BCG has failed in reducing the global TB burden, despite its widespread usage.
Interestingly, BCG protects children from TB, indicating that it has sufficient
antigenic repertoire to protect against Mtb. In contrast, failure to protect adults,
suggests BCG inability to generate long-lasting immunological memory. Further,
protection rendered by BCG against pulmonary TB in adults is highly inconsis-
tent, varying from 0% to 85% (Andersen and Doherty, Nat Rev Microbiol
3:656–62, 2005). Furthermore, its efficacy is least in TB-endemic countries.
Studies conducted in Malawi, India, and other endemic countries have concluded
that BCG induces inadequate protection. The probable reasons suggested are the
G. Kaur · J. N. Agrewala (*)

Immunology Laboratory, Institute of Microbial Technology, Chandigarh, India
Centre for Biomedical Engineering, Indian Institute of Technology-Ropar, Rupnagar, India
e-mail: jagrewala@iitrpr.ac.in
D. K. Das · S. Singh · J. Khan · M. Sajid · H. Bashir · M. Aqdas · S. Negi
Immunology Laboratory, Institute of Microbial Technology, Chandigarh, India
U. Gowthaman
Department of Laboratory Medicine, Immunobiology and Orthopedics,
Yale University School of Medicine, New Haven, CT, USA

https://doi.org/10.1007/978-981-32-9413-4_21
376 G. Kaur et al.
interference by nontuberculous mycobacteria (NTM) in antigen processing and

presentation of antigen-presenting cells (APCs), latent TB infection (LTBI), and
high prevalence of helminth infestation. There are currently 12 vaccine
candidates in clinical trials, including recombinant BCG vaccines, attenuated
Mtb strains, recombinant viral-vectored platforms, protein/adjuvant
combinations, and mycobacterial extracts (WHO, Global tuberculosis report,
2017). However, the whole-cell vaccine candidates may encounter same problem
in TB-endemic population, as mentioned above for BCG. Therefore, radically
novel strategies of vaccination against Mtb are urgently needed. Peptide vaccines
can be a possible alternative to overcome the failures of BCG because it
comprises of epitopes that can directly bind to MHC molecules, circumventing
the interference of NTM and latent Mtb in antigen processing. Furthermore,
epitopes from multiple stages like LTBI, active, chronic, and drug-resistant Mtb
can be selected. In essence, peptide-based prophylactic and therapeutic vaccines
represent a promising future strategy for efficient management of TB.
Keywords
Mycobacterium tuberculosis · Immune response · Vaccines · Peptide vaccines
Abbreviations
APCs Antigen-presenting cells

DCs Dendritic cells
DIM Phthiocerol dimycocerosates
Hly Listeriolysin
LTBI Latent TB infection
Mtb Mycobacterium tuberculosis
NTM Nontuberculous mycobacteria
PBMCs Peripheral blood mononuclear cells
pfo Perfringolysin
SNP Single nucleotide polymorphisms
TB Tuberculosis
Tregs T regulatory cells
21.1 Vaccine Development
Vaccination is considered as one of the cornerstones in the history of modern

medicine (Plotkin and Plotkin 2011). In case of various medical interventions, this
is the most widely implemented field that has gained considerable attention world-
wide. In the USA alone, vaccines have protected 100 million people from different
21 Tuberculosis Vaccine: Past Experiences and Future Prospects 377
diseases (Van Panhuis et al. 2013). Further, about 2.5 million deaths annually are
prevented globally (WHO 2013). Vaccines are quite important for protecting present
and future generations. Well-planned vaccination schedules have helped to save
hundreds of millions of people from death, apart from savings in public health
expenditures. An effective vaccine should be able to provide lifelong immunity
against a particular pathogen with no adverse side effects. Vaccines prime our
immune system by exposing an individual to a nonlethal, milder version of the
pathogen’s antigens in order to develop memory T cells, without causing disease.
Ideally, a vaccine is made of pathogen-derived components that are critical for
induction of protective immunity. Consequently, vaccines are composed of either
an attenuated or killed version of the bacteria, inactivated bacterial toxins, subunits
of the pathogen, or peptides that mimic naturally occurring proteins from pathogens
(Table 21.1).
21.2 BCG: The Only Available Vaccine Against TB
BCG is the only successful vaccine developed so far against TB infection. Despite
its widespread use, BCG delivers only minimal protection and has failed to
eradicate or reduce TB. BCG is derived from Mycobacterium bovis, a bovine
mycobacterium passaged 230 times in the Pasteur laboratory in Lyon by Albert
Calmette and Guérin between 1908 and its first human use in 1921. First adminis-
tration of BCG vaccine in humans was done by Benjamin Weill-Halle assisted by
Raymond Turpin at the Charité Hospital, Paris. On July 18, 1921, a healthy infant
was immunized with BCG whose mother died immediately after his birth because
of TB. BCG was fed orally in a spoon, followed by ingestion of milk. The child
remained healthy and showed no signs of TB. Furthermore, 114,000 infants were
vaccinated with BCG from 1924 to 1928 and no complications were observed
(Luca and Mihaescu 2013). Similar trials were conducted in Europe, Africa, South
America, and Asia. Earlier, BCG was given orally but now WHO recommends
intradermal route of vaccination, as it allows more consistent dosing. Other reason
can be that the efficacy of BCG reduces one- to two-fold on exposure to gastric
enzymes and pH (Tacquet et al. 1962). Moreover, it has been shown that intrader-
mal vaccination reduces the risk of TB by 50%. Interestingly, BCG not only
protects TB; rather it has also shown good results against leprosy (Orege et al.
1993). Intriguingly, BCG has exhibited quite convincing results as an immuno-
therapeutic agent against bladder cancer. It has since then become the most widely
used vaccine in human history, with over 3 billion doses administered. Currently,
more than 100 million children are annually administered BCG within hours or
days of birth. The protective efficacy of BCG is highest (85%) in TB non-endemic
population, whereas it is lowest (0%) in TB-endemic regions. Further, it protects
the childhood but not the adult manifestation of TB. Nonetheless, due to the overall
variability in its efficacy, BCG remains the most controversial vaccine.
378 G. Kaur et al.
Table 21.1 Outline of the development of human vaccines (Plotkin 2014)

Killed whole Purified proteins or
Live attenuated organisms polysaccharides Genetically engineered
Eighteenth century
Smallpox (1798)
Nineteenth century
Rabies (1885) Typhoid (1896)
Cholera (1896)
Plague (1897)
Early twentieth century, first half
Tuberculosis Pertussis (1926) Diphtheria toxoid (1923)
(bacillus Calmette-
Guérin) (1927)
Yellow fever (1935) Influenza Tetanus toxoid (1926)
(1936)
Rickettsia
(1938)
Twentieth century, second half
Polio (oral) (1963) Polio (injected) Anthrax secreted proteins Hepatitis B surface
(1955) (1970) antigen recombinant
(1986)
Measles (1963) Rabies (cell Meningococcus Lyme OspA (1998)
culture) (1980) polysaccharide (1974)
Mumps (1967) Japanese Cholera (recombinant
encephalitis toxin B) (1993)
(mouse brain)
(1992)
Rubella (1969) Tick-borne Pneumococcus
encephalitis polysaccharide (1977)
(1981)
Adenovirus (1980) Hepatitis A
(1996)
Typhoid Cholera Haemophilus influenzae
(SalmonellaTY21a) (WC-rBS) type B polysaccharide
(1989) (1991) (1985)
Varicella (1995) Meningococcal H. influenzae type b
conjugate conjugate (1987)
(group C)
(1999)
Rotavirus Japanese Typhoid
reassortants (1999) encephalitis (Vi) polysaccharide
(2009) (Vero (1994)
cell)
Cholera (attenuated) Cholera Acellular pertussis (1996)
(1994) (WC only)
(2009)
Cold-adapted Vaxchora for Hepatitis B (plasma
influenza (1999) the prevention derived) (1981)
of cholera
(2016)
(continued)

Killed whole Purified proteins or
Live attenuated organisms polysaccharides Genetically engineered
Twenty-first century
Rotavirus Pneumococcal conjugates Human papillomavirus
(attenuated and new (heptavalent) (2000) recombinant
reassortants) (2006) (quadrivalent) (2006)
Zoster (2006) Meningococcal Human papillomavirus
conjugates (quadrivalent) recombinant (bivalent)
(2005) (2009)
Pneumococcal conjugates Meningococcal group
(13-valent) (2010) B proteins (2013)
VSV-EBOV vaccine Dengvaxia live
against Ebola (2014) attenuated tetravalent
chimeric vaccine for
Dengue (2015)
Malaria vaccine, Shingrix (recombinant
P. falciparum protein zoster vaccine) to
fused with hepatitis B prevent shingles
surface antigen (2015) (2017)
Injectable influenza Heplisav-B, the new
vaccine, Fluad (2015) hepatitis B vaccine
(2017)
21.3 Modulation of Immunity by BCG
Macrophages, dendritic cells, and neutrophils are the major cells of immune system
and first line of defense to combat Mtb. Upon vaccination, epidermal macrophages
interact with BCG through the pattern recognition receptors (PRRs), such as toll-like
receptor 2/4 (TLR2/4), macrophage-inducible Ca2+-dependent lectin (MINCLE),
complement receptor 3 (CR3), and mannose receptor (MR) present on their surface
(de Koning et al. 2012; Bugarcic et al. 2008). In contrast, alveolar macrophages
interact with Mtb.
BCG provokes antigen-presenting cells (APCs) to secrete cytokines, chemokines
and recruit various cells of the immune system. The APCs process and present BCG
antigens to T cells. Activated CD4 T cells then release cytokines such as IFN-γ, IL-2,
and TNF-α that participate in protection against Mtb infection. IFN-γ-secreting CD4
T cells express CD45RA CCR7+/ CD62L phenotypic markers, indicating cen-
tral and effector memory cells in BCG-vaccinated donors (Li et al. 2011). Apart from
CD4 T cells, NK and γδ T cells produce IFN-γ on BCG vaccination (Ritz et al.
2012).
380 G. Kaur et al.
21.4 Activation of Innate Immunity by BCG
BCG activates dendritic cells (DCs) and macrophages mainly through TLR-2 and
TLR-4 by signaling cascade involving MyD88 adaptor molecule, which recruits
IRAK, TRAF6, and MAPK, eventually activating NF-κB. This leads to their
maturation and activation. This results in the release of proinflammatory cytokines
IL-12, TNF-α, and IL-1β (Takeda and Akira 2004). Various chemokines, matrix
metalloproteinases (MMPs), and adhesion molecules ICAM-1, VCAM-1,
p-selectins, L-selectins (CD62L), and E-selectins are upregulated (Kupper and
Fuhlbrigge 2004).
21.5 Activation of Adaptive Immunity by BCG
Th1 immunity is crucial for protection against Mtb. BCG vaccination in newborn is
reported to elicit Th1 cells that show effector memory phenotype
(CD45RA CCR7 CD27+) and release mainly IFN-γ, while central memory cells
secrete chiefly IL-2 (Soares et al. 2008). Further, recent studies have illustrated the
potent role of Th17 cells in protection against Mtb (Khader and Cooper 2008).
Furthermore, CD8 T cells play an important role in protection against TB. BCG
taken up by APCs is presented by DCs or macrophages in context with MHC class I
molecules to CD8 T cells. MHC-I-deficient (β2m / ) mice immunized with BCG
survived upon exposure to Mtb, indicating the importance of CD8 T cell immunity
(Flynn et al. 1992). Further, protective CD8 T cell response elicited by BCG in
newborn children was attributed to either cytotoxic degranulation or IFN-γ cytokine
secretion (Murray et al. 2006). Although BCG-induced CD4 T cells are more crucial
than CD8 T cells, both coordinate with each other to protect against Mtb. Th1
response generated against Mtb can be suppressed by Th2 cells. Thus, a predomi-
nance of Th1 cells is essential for protection against Mtb infection (Rook et al. 2004).
21.6 Reasons for Failure of BCG Vaccine
The Chingleput trial in India has established 0% protective efficiency of BCG

(Tuberculosis Research Centre) (Datta et al. 1999). Results have shown that the
efficacy of BCG lasts up to 15 years in the United Kingdom (Sterne et al. 1998),
30–40 years in Norway (Nguipdop-Djomo et al. 2016), and even higher up to
50–60 years in Alaska (Aronson et al. 2004). Exposure to nontuberculous
mycobacteria (NTMs) (Chiang and Riley 2005), genetically varied human popula-
tion (Brosch et al. 2007), different BCG strains (Osborn 1983), helminth infestation,
and exposure to nontuberculous mycobacteria (NTM) are among the main causes of
variable BCG efficacy. Despite its wide usage, BCG has not adequately reduced the
global TB burden. Due to its inconsistent performance across the globe, BCG
remains a debatable vaccine.
Fig. 21.1 Different BCG strains used globally and incidence of TB in the region (Ritz and Curtis
2009). Each circle represents group of countries. The outer circle signifies countries with a TB
incidence of >100/100,000 individuals; middle circle represents 40–100/100,000 and the inner
circle represents <40/100,000. The numbers in the doughnut graph symbolize the number of
countries within that particular category. Mixed means countries that use 2 or > 2 strains of
BCG. “Local BCG” implies to those countries, which employ indigenously produced BCG
Several reasons have been attributed for the variable efficacy of BCG vaccine
worldwide:
(i) Variation in BCG strains: The inconsistency of BCG vaccine might be

attributed to the variability in the seed strains produced/cultured by various
countries (Brosch et al. 2007). The original attenuated BCG was kept in running
cultures for several years until seed lots were preserved and stored. These
lyophilized strains were then distributed to various countries (Dockrell and
Smith 2017). Now with improved sequencing and omics technologies, genetic
and proteomic variations among these strains have been proved (Abdallah et al.
2015). These variations may be due to single nucleotide polymorphisms (SNP),
duplication, and deletions at genetic levels. Furthermore, some countries have
used more than one strain in their immunization program, which has added
complications in the variability of results (Ritz and Curtis 2009). A majority of
countries (105 of 188 countries) have used more than one strain of BCG in the
same country (Fig. 21.1). Genomic comparison and analysis of 13 different
BCG strains used for vaccination have shown a greater degree of variation in the
T cell epitopes due to higher deletion rates among them (Zhang et al. 2013).
(ii) Interference of nontuberculous mycobacteria (NTM): BCG protects neonates
against pulmonary TB but it fails to protect adults (Dark 1978). This is due to
inability of BCG to generate long-lasting memory T cells. This is evident from a
382 G. Kaur et al.
Fig. 21.2 (a) Masking hypothesis: exposure to NTMs at an early age increased the individual’s
basal immunity against the mycobacteria (blue line). The same individual when vaccinated with
BCG at a later age (red line), BCG-induced immunity could not be further enhanced due to masking
of immune response by prior exposure to NTMs. (b) Blocking hypothesis: Pre-exposure to NTMs at
an earlier age increased the individual’s immunity against mycobacteria. The same individual when
vaccinated with BCG, the immune response generated against NTM obstructed BCG replication
and therefore prevented the optimum exposure of BCG antigens to the immune system. Therefore,
the person is not protected from Mtb infection, as noted in the case of TB-endemic population. In
contrast, in the absence of NTMs, BCG generates sufficient immunity to protect against Mtb, as
observed in TB non-endemic regions
study where mice that have been given NTMs by oral gavage following BCG
vaccination showed decreased long-lasting protection (Flaherty et al. 2006). The
possible reason suggested was that in TB-endemic regions like India, BCG
efficacy was lower because of exposure to NTM (Palmer and Long 1966).
Nontuberculous mycobacteria hinders the proliferation of BCG. Consequently,
the immune system is not optimally exposed to BCG antigens and therefore fails
to elicit adequate immunity. Majorly, two hypothesis has been attributed to this
cause (Fig. 21.2):
Masking hypothesis. According to this hypothesis, exposure of human to NTMs

generates a baseline immunity. Thus, the baseline immunity already
generated by NTMs masks the BCG-induced response (Fig. 21.2a). A
clinical study encompassing school children from two entirely different
populations, viz., UK (a country with lowest TB incidence and with highest
BCG success) and Malawi (a region in which BCG vaccination has shown no
effect against pulmonary TB), was reported. Subjects from UK had a low
baseline cellular immunity to mycobacteria, which was increased upon BCG
immunization. On the contrary, subjects from Malawi had a higher level of
basal cellular immunity against mycobacteria, due to pre-exposure of NTMs
in this region, and thus the immunity was not enhanced by BCG (Black et al.
2002).
Blocking hypothesis. In this case, the preexisting immune response generated
due to NTMs prevented the replication of BCG (Fig. 21.2b). Mice sensitized
with environmental NTMs isolated from soil and sputum samples from
Malawi hampered the growth of BCG without any effect on TB incidence
(Fine et al. 2001).
Studies have also suggested that BCG itself hampers antigen processing and
presentation of its own antigens, consequently generating an inadequate immune
response (Singh et al. 2006). BCG evades lysosomal fusion in macrophages (Ullrich
et al. 2000). This results in reduction of antigen processing and downregulating the
expression of MHC-II molecules. These macrophages have lower capability to
activate Th1 immunity, which is a prerequisite for protection against Mtb. Further,
NTMs can enhance T regulatory cell (Tregs) expansion and recruitment, thus
retarding BCG efficacy (Ho et al. 2010).
(iii) Helminth infestation: Another important cause for BCG failure in the
TB-endemic regions is the prevalence of helminth infestation. Helminth
enhances the Th2-type immunity and higher IgE profile and increases the
level of Treg cells (Elias et al. 2007). Clinical study has proved that the
infection with Ascaris lumbricoides can hamper the generation of a proper
Th1 immunity (Van Soelen et al. 2012). Similarly, Strongyloides stercoralis
infection skews the immunity toward Th2 type (Carvalho and Da Fonseca
Porto 2004). These worms interfere with the performance of BCG. Infection
with Schistosoma mansoni results in an elevated level of IL-4/IFN-γ ratio
(Zwingenberger et al. 1991). A lower IL-4/IFN-γ ratio in the bronchoalveolar
lavage and pleural fluid has been correlated with a better immunity against TB
(Ashenafi et al. 2014). However, it has shown that helminth infestations do not
reduce the efficacy of BCG by increasing the Th2 cells but by enhancing
TGF-β production (Elias et al. 2008).
(iv) Route of administration: The route of administration of BCG is disputed.
Originally, BCG was administered orally and a study showed that oral route
involves lesser pathology and similar level of protection (Dockrell and Smith
2017). While dose responsiveness of BCG vaccination is not prominent in
384 G. Kaur et al.
subcutaneous route of immunization, intratracheal route offers better dose

responsiveness and better reduction in Mtb CFU count in lungs (Aguilo et al.
2014). Further, advantage in intratracheal and intranasal BCG vaccination
includes elicitation of T effector memory (TEM) and T resident memory (TRM)
cells in the lung tissue. Contrary to this, subcutaneous and intradermal routes of
BCG immunization fail to offer memory T cell response (Verreck et al. 2017).
(v) Batch to batch variation of BCG strain: The culturing of BCG involves
various steps and thus differences have been observed in the growth pattern
among batches. The batches respond differently toward sensitivity against
isoniazid, which correlates with the immunity they provide in mice. Further,
randomized clinical trials in neonates have supported dissimilarity in the
immune response (Biering-Sørensen et al. 2015).
(vi) BCG triggers the generation of Tregs and Th2 cells: Another dark side of BCG
vaccine is that it can directly dampen anti-TB response by generating Tregs
and Th2 cells (Lagranderie and Guyonvarc’h 2014). The dose of BCG vaccine
influences the type of T cell response generated. A lower dose of BCG
generates Th1 cells, while a higher dose of BCG leads to mixed generation
of Th1 and Th2 cells (Power et al. 1998).
(vii) Nonspecific activity of BCG: BCG shows promising results in bladder cancer,
melanoma cancer, asthma, leprosy therapies, and probably against Buruli ulcer
(Fuge et al. 2015; El-Zein et al. 2017). Consequently, this casts a doubt on
BCG reliability as a vaccine. Rather it works as an immunomodulator to
bolster host immunity against array of diseases (Fig. 21.3). This can be due
to the fact that the generation of nonspecific immune response by BCG may be
due to the activation of mononuclear phagocytic system via PAMPS like
TLR-2, TLR-4, and TLR-8 signaling, consequently initiating a cascade of
inflammatory responses and thereby recruiting inflammatory cells to the site
of infection. Further, it provides feedback signals for maturation of DCs,
macrophages, and neutrophils.
Fig. 21.3 Diverse roles of

BCG. BCG not only protects
against TB but also from
cancers, Buruli ulcer, asthma,
and leprosy. This activity of
BCG may be due to its
immunomodulatory activity,
since it can trigger cascade of
signaling events through
TLR-2, TLR-4, and TLR-8
21.7 Prospects for New TB Vaccine
For complete eradication of TB, an effective vaccine is urgently needed. Despite the
fact that TB can be cured, 10 million people developed TB disease in 2017: 5.8
million men, 3.2 million women, and 1.0 million children. Globally, 3.5% of new
TB cases and 18% of previously treated cases had MDR/RR-TB (WHO 2018).
Therefore, it is a daunting task to reduce TB burden. Currently, it is important to
develop drugs or vaccines to control the emergence and spread of drug-resistant
TB. The treatment for drug-resistant TB is not only costly but has devastating toxic
effects. The new generation of vaccines being developed should target TB and its
drug-resistant forms. An effective TB vaccine may be prophylactic and/or therapeu-
tic. An ideal therapeutic vaccine should shorten the dose and duration of treatment
and improve the potency of drugs. A prophylactic vaccine would be safer in pre- or
postexposure to Mtb infection and effective in averting primary and latent infection
and reactivation of latent forms. This can be achieved by two ways, either replacing
BCG with an alternative live mycobacterial vaccine or boosting the efficacy of BCG
with the subunit vaccines. The second approach may be to combine both approaches
by priming with a novel live mycobacterial vaccine and boosting with a subunit
vaccine. During last few years, there has been a tremendous pressure on researchers
to develop a safe and effective vaccine against TB. Some of the vaccine candidates
have entered various stages of clinical trials.
21.8 Recombinant BCG Vaccines (rBCG)
Importantly, an ideal immune phenotype of protection should be taken into consid-

eration while designing an effective vaccine. A successful vaccine should generate
appropriate and adequate immunity, enduring memory T cells and protection from
the pathogen on subsequent encounter. In mouse model, currently accepted memory
phenotypes are CD44hiCD62LloCCR7lo for effector memory T cells,
CD44hiCD62LhiCCR7hi for central memory T cells, and CD69hiCD103hi CD62Llo
for resident memory T cells (Henao-Tamayo et al. 2010). IFN-γ-secreting CD4 T
cells are one of the major targets of TB vaccine research. IFN-γ-producing CD8 T
cells too play a crucial role in protection against Mtb (Flynn et al. 1993). CD8 T cells
play a significant role in controlling latent TB infection (LTBI) (van Pinxteren et al.
2000). Potent rBCG vaccines should induce a balanced number of Mtb-specific CD4
and CD8 T cells. Studies have shown that desired memory T cells should have
polyfunctional characteristics, i.e., ability to secrete IFN-γ, tumor necrosis factor
(TNF)-α, and IL-2 to provide protection against Mtb infection (Bouneaud et al.
2005). Combination of biomarkers for human memory T cells with effector pheno-
type includes CD45RAhiCD45RO CCR7 and for central memory T cells
CD45RAhi/loCD45RO CCR7+. Long-lasting memory T cells expressing CD127
(IL-7 receptor) are associated with protection against Mtb infection (Seder and
Ahmed 2003). Beyond doubt, BCG has imparted significant protection against
386 G. Kaur et al.
Mtb in the Western population and childhood manifestation of the disease globally.
BCG vaccine is cost-effective, stable, and easy to manufacture. It has an ability to
express foreign antigens in their functional form. These attributes have driven the
development of various forms of rBCG. We discuss here some potential rBCG
vaccine candidates and their ability to protect against TB.
21.9 Different Strategies Used in the Development of rBCG
Over the past several years, various strategies have been strived in the advancement
of rBCG (Table 21.2). One of the major strategies adopted is the overexpression of
immunodominant antigens of Mtb such as antigen 85 (Ag85) complex and
α-crystalline protein (HspX). Other strategy is the reintroduction of antigens such
as CFP-10, ESAT-6, INV and MPT64, HBHA, PE/PPE family, ApA, MDP1,
MPT64, PepA, Rv0111, and Rv2520c that might have been deleted during
Table 21.2 Description of strains and antigens/modifications used in the rBCG development
Vaccine Strain Description Model References
rBCG30 BCG Overexpression of Ag85B Human Hoft et al.
tice (2008)
rBCG-Ag85A BCG Overexpression of Ag85A Monkey Sugawara
Tokyo et al. (2009)
rBCG-85C BCG Overexpression of Ag85C Guinea Jain et al.
Danish pigs (2008)
rBCG:X BCG Expresses HspX protein Mice Shi et al.
Danish (2010)
rBCG:PhspX- BCG Expresses Ag85B under control of Mice Kong et al.
85B Pasteur promoter HspX (2011)
rBCG-AE BCG Expresses fusion protein Ag85A- Mice Deng et al.
China ESAT-6 (2014)
rBCG-Aeras BCG Endosome escape activity with Monkey Rahman
403 Danish overexpression of Ag85A, Ag85B, et al. (2012)
TB10.4
rBCGΔureC: BCG Endosome escape activity Mice Desel et al.
hlyC Parague (2011)
BCG:RDI BCG Reintroduction of RD1 gene regions Mice Pym et al.
Pasteur (2003)
rBCG-E6 BCG Reintroduction of ESAT-6 Guinea Dey et al.
Danish pigs (2009)
rBCG:Ag85B- BCG Co-expresses Ag85B, ESAT-6, and Mice Lu et al.
ESAT-6- Danish Rv2608 (2012)
Rv2608
rBCG-AMM BCG Expresses Ag85B-MPT64-Mtb8.4 Mice Qie et al.
Danish (2009)
Pro-apoptotic BCG Deletion nlaA gene that inhibits Mice Chattergoon
BCG tice apoptosis et al. (2000)
attenuation procedure of BCG but are crucial in imparting protective immunity (Jain
et al. 2008). Ag85A (Rv3804c), Ag85B (Rv1886c), and Ag85C (Rv0129c) are
important antigens from Ag85 complex used to develop rBCG vaccines. These
proteins have mycolyl-transferase activity that is necessary for the synthesis of
mycobacterial cell wall (Belisle et al. 1997). Horwitz et al. have developed
rBCG30 that overexpresses Ag85B. Ag85A is the most abundant secretory protein
of Mtb and potent inducer of IFN-γ production and Th1 immunity (Horwitz et al.
2000). In guinea pigs, rBCG30 showed increased protection against Mtb, as com-
pared to wild-type BCG. Phase I clinical trials with rBCG30 in around 30 healthy
individual upshots increased immunogenicity in terms of central and effector mem-
ory CD4 T cells and CD8 T cells, as compared to parental BCG (Hoft et al. 2008).
Unfortunately, the outcome of rBCG30 clinical trial did not meet the expected
immunogenicity and further testing was discontinued. Tullius et al. developed a
mutant strain rBCG(mbt)30 that was unable to produce mycobactin and exoquelin
molecules required for iron acquisition. In another strategy, rBCG pantothenate
auxotroph rBCG(PanCD)30 was designed (Tullius et al. 2008). Both these vaccines
showed higher degree of attenuation than BCG and instigated potent protective
immunity in guinea pigs. These vaccine candidates may provide better and safer
protection. Ag85A strongly induced T cells to secrete IL-2 and IFN-γ (Lozes et al.
1997). rBCG:Ag85A-vaccinated mice and guinea pigs provided better protection
than BCG against Mtb in pulmonary and spleen infection (Sugawara et al. 2007).
Further, this vaccine was investigated in rhesus monkeys (Macaca mulatta). rBCG:
Ag85A induced greater efficacy than BCG Tokyo strain. rBCG:Ag85C-inoculated
guinea pigs imparted better protection, as evidenced by reduced lung infection and
granuloma formation (Jain et al. 2008).
Combination of two or more immunodominant antigens of Mtb as fusion protein
in rBCG was hypothesized to attain better efficacy. Wang et al. developed three
vaccines rBCG:Ag85A, rBCG:Ag85B, and rBCG:Ag85AB. The rBCG expressing
fusion protein (Ag85A-Ag85B) exhibited greater protection than rBCG:Ag85A and
rBCG:Ag85B alone (Wang et al. 2012). HspX, known as α-crystalline, is a 16 kDa
heat-shock protein that is potentially used in the development of rBCG. Mtb
produces HspX protein in high amounts during latent and persistence metabolic
conditions. Shi et al. developed a rBCG:X vaccine that overexpresses HspX. rBCG:
X provided greater efficacy and long-lasting protection against Mtb than parental
BCG. rBCG:X induced high level of IFN-γ and reduced mycobacterium load in the
lungs and other tissues (Shi et al. 2010). Kong et al. generated rBCG:HspX-Ag85B
that overexpressed Ag85B under the control of HspX promoter. This vaccine
induced IFN-γ and T cell proliferation (Kong et al. 2011).
BCG favorably resides inside endosomes of macrophages and DCs. This locali-
zation supports the presentation of BCG antigens in association with MHC-II to CD4
T cells, resulting in their activation, proliferation, and differentiation. CD8 T cells are
activated when antigen is presented in context with MHC-I. Thus to activate Mtb-
specific CD8 T cells, BCG must gain access from endosome into cytosol of the host
cells. In this context, Kaufmann et al. developed rBCGΔUreC:Hly+ (VPM1002)
vaccine (Nieuwenhuizen et al. 2017). It secretes listeriolysin (Hly) and contains an
388 G. Kaur et al.
inactivated urease C gene to ensure optimal Hly activity in an acidic phagosomal

environment (Albert et al. 1998). Listeriolysin of rBCGΔUreC:Hly+ punches holes
into phagosomal membrane and enables BCG to escape from endosome into the
cytosol of cells. Further, it induces apoptosis and autophagy that facilitates the cross-
presentation of antigens to CD8 T cells. Further, robust activation of Th1 cells and
Th17 cells was noticed. Furthermore, it successfully expands central memory CD4 T
cells and CD8 T cells. In essence, rBCGΔUreC:Hly+ provokes immunity that is
crucial for protection against Mtb. Moreover, the vaccine is cleared more rapidly
from tissues than BCG, thus resulting in reduced persistence in the host. Sun et al.
developed a rBCG-pfo that expresses perfringolysin in a pH-independent manner
with overexpression of Ag85A, Ag85B, and TB10.4. Here, BCG escapes endosome
using perfringolysin. This vaccine showed superior protection over rBCG30 with
enhanced protection and safety of the endosome escape construct (Fig. 21.4)
(Portnoy et al. 1992). Jacob et al. proposed a pro-apoptotic vaccine by deleting
nlaA genes (Rv3238c) that inhibits apoptosis of infected host cells. rBCG vaccine
that promotes caspase-dependent apoptosis of host cells leads to the cross-priming of
CD8 T cells. Caspase-mediated apoptosis is very well known for the induction of
powerful cellular immunity (Chattergoon et al. 2000).
Reintroduction of genes that were deleted during the attenuation procedure of
BCG is also one of the important strategies for rBCG development. Virulence region
RD1 is absent in all sub-strains of BCG, but present in virulent mycobacteria
(Brosch et al. 2000). Cole et al. made an effort to improve the efficacy of the vaccine
by reintroduction of some lost genes into BCG. This strategy used the collection of T
cell epitopes, which were lost during attenuation of BCG. These gene regions
include RD1 locus, which encodes immunodominant proteins CFP-10 (Rv3874),
ESAT-6, INV (Rv1474), and MPT64.
Cytokines mediate important roles in regulation of homeostasis of naive and
memory T cells and their response to pathogens. Introducing genes encoding
mammalian cytokines, or cytokine antagonists such as the latency activating peptide
(LAP) that regulates transforming growth factor-β (TGF-β), can increase immuno-
genicity of BCG (Wilkinson et al. 2000; Slobbe et al. 1999). However, the limitation
of this strategy is that enhanced Th1 response may cause clearance of vaccine and
therefore will reduce memory response. Further, memory enhancing cytokines like
IL-7 and IL-15, play a dominant role in the generation and homeostasis of memory T
cells (Singh et al. 2010). Numerous studies have shown the adjuvant nature of IL-7
and IL-15 and their role in the generation and sustenance of memory T cells (Singh
et al. 2011; Nanjappa et al. 2008). Therefore, supplementing vaccines with IL-7 and
IL-15 may enhance the long-term sustenance of memory T cells. Interestingly,
substantial increase in the enduring memory T cells in mouse against Mtb infection
by supplementing BCG with rIL-7 and rIL-15 was observed. Further, it significantly
reduced the mycobacterial load and alleviated the pathology in the lungs, thus
providing better protection than BCG (Singh et al. 2010). Similarly, they showed
that cytokines like IL-1, IL-6, and TNF-α bolstered BCG potency by generating
long-lasting protective memory T cell response (Singh et al. 2011). More recently,
rBCG expressing Ag85B-IL-7 fusion protein was developed, which increased the
Fig. 21.4 Mechanisms of the immune responses generated by rBCGΔUreC:Hly+. (a)

rBCGΔUreC:Hly+ (with a deletion of UreC and expression of listeriolysin:Hly+) or rBCG-pfo
(expressing pH-independent perfringolysin) enable the vaccine to form pores in the early
phagosome that facilitate the leakage of rBCG into the cytoplasm of host cells. This results in
strong presentation of peptide in association with MHC class I molecules and cross-priming of CD8
T cells, generation of Th1 cells and Th17 cells, and induction of apoptosis. (b) Cross-priming and
enhanced apoptosis increase the uptake of rBCG antigens in the form of vesicles by DCs and
generates CD4 T cell and CD8 T cell immunity. (c) CD8 T cells coordinate with CD4 T cells in
eliminating Mtb. CD8 T cell utilizes its perforin, granzyme, and TNF-α to lyse Mtb-infected
macrophages, whereas CD4 Th1 cells produce IFN-γ to activate macrophages to kill Mtb
frequency of IL-17A+ γδ T cells and enhanced Th1 response against Mtb (Hatano
et al. 2016). Thereby, developing a recombinant BCG expressing memory enhanc-
ing cytokines can be an effective strategy to successfully improve the efficacy of
BCG in protecting not only childhood but also adult manifestation of TB.
ChAdOx1.PPE15 is a recombinant, replication-deficient chimpanzee adenovirus,
expressing the mycobacterial PPE15 protein of Mtb (WHO 2017). This candidate is
part of the overall UOXF TB vaccine program to work toward a BCG booster
vaccination regimen in adults and is being evaluated in guinea pigs for improved
protective efficacy.
CysVac2 is a fusion protein vaccine composed of Ag85B and CysD, a major
component of the sulfate activation pathway of Mtb. It was designed to target both
active and chronic phase of infection. Preclinical experiments in mice demonstrated
that the fusion protein provides strong protection when combined with the novel
adjuvant MPL/DDA and induced the generation of multifunctional CD4 T cells
390 G. Kaur et al.
expressing IFN-γ, IL-2, and TNF, as well as double-positive IFN-γ+TNF+ and

IL-2+TNF-α+ CD4 T cells in the lung (Counoupas et al. 2016). The vaccine was
particularly effective at controlling late-stage infection, a major problem in case of
BCG vaccination. The vaccine is being tested in guinea pigs to strengthen its case for
deployment in clinical trials.
MVA85A is a recombinant vaccine expressing Ag85A in modified vaccinia
Ankara virus (McShane et al. 2005). In rhesus macaques, the vaccine delivered by
aerosol route showed a high immunogenic effect (White et al. 2013). This approach
generated strong antigen-specific responses in the lungs and was well tolerated.
Moreover, this route of immunization did not generated antibodies to the viral
vehicle. In phase I studies, a well-tolerated and immunogenic effect with only
minor adverse reaction was noticed in HIV-infected adults in Senegal (Dieye et al.
2013). This was the first virally vectored vaccine to be tested in humans delivered
through aerosol route. Furthermore, in a randomized phase I trial, MVA85 adminis-
tration via intramuscular and intradermal routes in 24 healthy human volunteers
proved safe and well tolerated. Both routes of immunization led to generation of
strong and sustained Ag-specific CD4 T cell responses. MV85A was well tolerated
and immunogenic in a phase IIb trials conducted on 2797 healthy infants who had
previously been vaccinated with BCG. However, the vaccine showed no significant
protection against TB infection (Tameris et al. 2014). The results from this study
suggested that CD4 T cells induced by MVA85A do not correlate with protection
against Mtb infection. MVA85A induces polyfunctional CD4 T cells expressing
IFN-γ, IL-2, TNF-α, and IL-17 (Satti et al. 2014; Scriba et al. 2010). Detailed future
studies will be required to determine whether the protective efficacy of MVA85A
can be improved.
Ad35/AERAS-402 is an adenovirus-vectored vaccine formed by non-replicating
adenovirus serotype 35 (Ad35), expressing fusion protein composed of three major
antigens of Mtb Ag85A, Ag85B, and TB10.4 (WHO 2014). It is primarily designed as a
booster vaccine. The vaccine was well tolerated and found to be safe in a phase I study
(Churchyard et al. 2015) [NCT Identifier 01017536]. Vaccination with AERAS-402
with MV85A booster showed increase in antigen-specific T cell responses and poly-
clonal CD8 T cells (IFN-γ+TNF-α+IL-2+) [NCT 01683773] (Sheehan et al. 2015). The
studies have shown the safety and immunogenicity of vaccine in healthy infants,
previously vaccinated with BCG (Kagina et al. 2014). Aerosolized delivery of
AERAS402 in rhesus macaques induced potent Ag85A/B-specific, cytokine-producing
effector CD4 T cell and CD8 T cell response. Unfortunately, no enhanced survival or
decrease in bacterial burden was observed (Darrah et al. 2014).
M. indicus pranii (MIP) is a progenitor of M. avium complex, sharing cross-
reactive antigens with Mtb and M. leprae. Studies in mouse and guinea pigs have
shown its prophylactic and therapeutic potential, resulting in a significant decrease in
bacillary burden, reduced pathology, and enhanced survival (Singh et al. 2017; Gupta
et al. 2012). Comparative genomic analysis of MIP has attributed its immunomodula-
tory properties to its high antigenic potential. Recently concluded multicentric clinical
trial in category-II TB patients further confirmed its immunotherapeutic role in
difficult to treat patients having advanced disease (Sharma et al. 2017). MIP was
found to be safe without any adverse effects. Significantly higher number of patients in
the MIP group showing sputum culture conversion, as early as 4 weeks after initiation
of therapy, suggests elimination of detectable bacilli, which may help in shortening the
treatment duration, indicating its role in the prevention of TB.
Although Mtb infects one-third of global population, only 5–10% develop active
TB and 90–95% remain protected. This fact depicts that individuals infected with
Mtb develop effective and enduring protective immunity, thereby suggesting that
growing bacterium may produce intracellular antigens in the macrophage that may
be crucial for protective immunity. Based on this rational, Sharma and Agrewala
developed a vaccine by culturing live Mtb in the macrophages to an extent so that the
bacilli release sufficient antigens. The mycobacterium was then killed by anti-TB
drugs and the preparation was made safe by gamma irradiation. The cells were
adoptively transferred in the Mtb-exposed animals. The results showed optimum
immunity and better protection than BCG (Sharma and Agrewala 2004). In future,
macrophage-based vaccine may generate same results as noticed in the case of DCs
used for successful treatment of cancer (Sabado and Bhardwaj 2010).
21.10 Peptide Vaccines
Peptide vaccines consist of epitopes capable of inducing both T cell- and B cell-
mediated immune responses. Peptides used in these vaccines are generally 20–30
amino acid sequences that are synthesized to form an immunogenic peptide mole-
cule representing the specific epitope of an antigen. The structural and proteolytic
stability of the synthetic peptides is further improved by linking them to adjuvants
that subsequently act as activators of the immune system. Peptides give an advantage
of selecting immunodominant CD4 T cell and CD8 T cell epitopes of the pathogen
and avoiding moieties that can provoke autoreactivity/autoimmunity. Peptides are
synthetic in nature and do not possess infectious material. Therefore, there is no
possibility of reverting to pathogenic status, a problem that is usually associated with
attenuated or mutated strains of pathogens. Conversely, peptide vaccines are often
weakly immunogenic and therefore require adjuvant for their immunogenicity.
Manufacturing of peptide vaccines is generally a safe and cost-effective option,
when compared to conventional vaccines. There are many peptide vaccines under
developmental stage for multiple diseases such as HIV, hepatitis C virus (HCV),
malaria, swine fever, influenza, anthrax, human papilloma virus (HPV), diabetes
mellitus, Alzheimer’s disease, and therapeutic anticancer vaccines. Majority of
candidate peptide vaccines are under phase I (270 studies) and phase II (224 studies)
stage of development. For instance, phase I clinical trials with GP2, a HER2/neu-
derived peptide, demonstrated that GP2-based vaccines are safe and effective in
stimulating peptide-specific immunity. The GP2 peptide vaccine is currently being
evaluated in a phase II trial against breast cancer (Clive et al. 2012). Only 12 studies
have progressed to phase III clinical stage and are being indicated for treatment of
multiple types of cancers (Li et al. 2014). However, the commercialization of a
successful peptide vaccine is still an unfulfilled dream.
392 G. Kaur et al.
21.11 Lipidated Promiscuous Peptide Vaccine (L91)
As mentioned above, there is an urgent demand for a radically different vaccine for
TB-endemic population, where BCG has totally failed (Datta et al. 1999). Thus, a
successful vaccine should meet the following attributes: (i) does not require exten-
sive antigen processing, (ii) not affected by the presence of preexisting antibodies/
immunity against mycobacteria or BCG, (iii) composed of appropriate CD4 and
CD8 T cell epitopes, and (iv) deliver suitable stimulatory signals for elicitation of
innate and adaptive immunity. Keeping these points in mind, Agrewala et al.
synthesized a chimeric lipidated peptide vaccine comprising of MHC-I and
MHC-II promiscuous epitopes selected from latent, early, and chronic stages of
Mtb conjugated to TLR-2 agonist Pam2Cys. This is a totally synthetic vaccine and
therefore can be used in AIDS and immunocompromised subjects. Most of the
peptides are poor immunogens. Therefore, the peptides were conjugated to Pam2Cys
to make it more immunogenic.
A peptide vaccine can be an effective and successful stratagem in TB-endemic
regions, since peptides do not require extensive antigen processing, as they can
directly bind to MHC molecules for being presented to T cell for their activation,
proliferation, and differentiation. Lipidated peptide vaccine has self-adjuvanting
property due to the presence of Pam2Cys and therefore does not require any
exogenous adjuvant. Pam2Cys binds to TLR-2 and induces local inflammation
and generates long-lasting protective T cell immunity against Mtb. The construct
can be targeted to DCs through TLR-2, which are copiously expressed on DCs. DCs
are the only APCs, capable of activating naïve T cells. Signaling through TLR2
triggers robust release of cytokines like IL-12, which induces protective Th1 immu-
nity. In addition, it stimulates nitric oxide (NO) release by macrophages that limit the
intracellular survival of Mtb (Thoma-Uszynski et al. 2001). Further, peptides have
no fear of obstruction in their processing by NTMs or antigen released by Mtb
present in latency inside the macrophages. Hence, lipidated promiscuous peptides do
not entail extensive processing and overcome the blocking effect of preexisting
antibodies in the sera of individuals of TB-endemic area. Furthermore, inclusion
of non-cross-reactive epitopes helps to evade the inappropriate immune responses
such as autoimmunity through cross-reactivity of pathogenic components with the
host proteins (Gowthaman et al. 2012). In human, HLA polymorphism renders
majority of peptides ineffective because they bind to only one or two HLA alleles.
On the other hand, promiscuous peptides permissively bind to several HLA alleles,
therefore overcoming HLA polymorphism. Promiscuous CD4 T cell epitopes were
identified from 16 kDa antigen (α-crystalline protein) of Mtb that provokes strong
Th1 immune response after being tested using PBMCs of individuals with diverse
HLA alleles (Agrewala et al. 1999). In addition, CD8 T cell epitopes were selected
from TB10.4 antigen of Mtb. Vaccination with the construct elicits enduring mem-
ory T cell response and provides significantly better protection than BCG against
Mtb. Furthermore, the chimera works as a prophylactic and therapeutic vaccine and
reduces the dose and duration of TB drug regimen (Rai et al. 2016). These findings
suggest a potent role of lipidated peptide vaccines in the prevention of disease,
particularly in TB-endemic areas.
21.12 TB Vaccine Candidates in Clinical Trials
The status of new TB vaccines in pipeline in various stages of development, as

published by WHO in August 2017, is shown in Fig. 21.5. There are 12 vaccines in
phase I, II, or III trials. Their main characteristics are summarized below. Ad5
Ag85A is a human recombinant replication-deficient adenovirus serotype 5 vaccine
vector expressing Ag85A (fbpA, Rv3804c, secreted antigen 85A,
mycolyltransferase 85A), an immunodominant antigen produced by all mycobacte-
rial species. The vaccine was evaluated in a phase I trial in 12 BCG-naive and
12 previously BCG-immunized, healthy Canadian adults, which demonstrated no
vaccine-related serious adverse events. The study showed enhanced CD4 T cell and
CD8 T cell immunity in previously BCG-vaccinated individuals compared to
BCG-naive volunteers, supporting its further clinical development as a booster
vaccine after BCG priming (Smaill and Xing 2014). The vaccine also showed
safe, immunogenic, and enhanced protection against Mtb challenge in murine,
bovine, and guinea pig models [NCT00800670]. BCG-primed guinea pigs when
intranasally boosted with AdAg85A showed significant enhancement in their sur-
vival rate following pulmonary Mtb challenge (Xing et al. 2009). A safety and
immunogenicity study of this vaccine by aerosol route has begun in
BCG-vaccinated healthy volunteers.
MTBVAC is a live attenuated Mtb strain with deleted phoP and fadD26 genes.
PhoP is a transcription factor that controls 2% of the genome of Mtb including
production of immunomodulatory cell wall lipids and ESAT-6 secretion (Gonzalo-
Asensio et al. 2008). Deletion of fadD26 leads to complete abrogation of synthesis of
virulence surface lipids phthiocerol dimycocerosates (DIM) (Camacho et al. 1999).
The main advantage of using attenuated live Mtb as vaccine is that many genetic
regions encoding important immunodominant antigens that are absent in BCG are
still present in attenuated Mtb. Moreover, chromosomal deletions in virulence genes
provide an assurance for safety and genetic stability. The primary target population
is newborns (as replacement for BCG) and secondary target includes adolescences
(booster with BCG). MTBVAC exhibits safety and bio-distribution profiles similar
Fig. 21.5 Flowchart showing TB vaccines in pipeline

394 G. Kaur et al.
to BCG and confers superior protection in preclinical studies (Arbues et al. 2013).
MTBVAC has been the first and only live attenuated Mtb vaccine approved to enter
into clinical trials. A first-in-human MTBVAC clinical trial was recently conducted
successfully in healthy adults in Lausanne (Switzerland) [NCT02013245]. In
September 2015, MTBVAC entered phase Ib trial in infants under the
South African Tuberculosis Vaccine Initiative (SATVI) program. Phase IIa trials
in both the target populations are expected to start in 2018. Highly attenuated
MTBVAC constructed with inactivation of an additional gene generated in exported
repeated protein (Erp) could be a potential TB vaccine candidate for use in a high-
risk immunosuppressive population (Solans et al. 2014).
ChAdOx185A-MVA85A is a simian adenovirus and MVA85A is a recombinant
pox virus vaccine, expressing antigen 85A. These candidate vaccines aim to gener-
ate a joint heterologous prime-boost regimen delivered through both systemic and
mucosal routes. MVA85A induces primarily CD4T cell responses and recombinant
adenoviral vectors induce primarily CD8 T cell responses. Intranasal administration
of ChAdOx1.85A induced strong immune responses in the lungs but failed to protect
animals against aerosol Mtb challenge. In contrast, ChAdOx1.85A followed by
MVA85A administered either mucosally or systemically induced strong immune
responses and was able to improve the protective efficacy of BCG (Stylianou et al.
2015). Phase I trial of ChAdOx185A by i.m. route in BCG-vaccinated adults in UK,
both alone and as part of a prime-boost strategy with MVA85A, has been completed
[WHO 2017, NCT01829490].
As per WHO report 2017, there are currently following nine vaccines in phase II
or phase III trials (Fig. 21.5). DAR-901 booster is a whole-cell vaccine, prepared
from nontuberculous mycobacteria (M. obuense). It is developed at the Geisel
School of Medicine, Dartmouth University, USA. DAR-901 represents broth-
grown, scalable variant of SRL172 (agar-grown). SRL172 is the only TB vaccine
which has showed reduction in the culture-confirmed Mtb in a phase III randomized
clinical trials and was found to be safe, well-tolerated, and immunogenic in humans.
A randomized controlled phase III trial in Tanzania showed protection against Mtb
upon boosting with SRL172 in HIV-infected adults, who had received BCG at birth
(Solans et al. 2014). A recent study showed that a DAR-901 booster is more effective
at providing protection against Mtb than a BCG booster (Lahey et al. 2016).
Hybrid 4/IC31 (AERAS-404) contains a fusion protein of Ag85B and TB10.4,
with IC31 as adjuvant. IC-31 is a two-component adjuvant comprising an 11-mer
antibacterial peptide (KLK) and a synthetic oligodeoxynucleotide (ODN1a), a
TLR-9 agonist (Aichinger et al. 2011). The vaccine candidate has been shown to
be immunogenic and protective in a preclinical animal model of TB. It is known to
induce persistent polyfunctional CD4 T cell responses in adults. A phase II study of
its ability to prevent infection in healthy adults has been completed (NCT02075203).
It is also in a phase I/II trial in infants.
H56:IC31 is a protein subunit vaccine comprised of fusion protein of Ag85B,
ESAT-6 (two of the Mtb antigens secreted in the acute phase of infection), and the
nutrient stress-induced antigen Rv2660c, formulated in IC31 adjuvant (WHO 2014).
In mice, H56:IC31 could control late-stage infection and reduced bacterial burden
more efficiently than BCG, 24 weeks postinfection. It can work both as preventive
and postexposure vaccine. As a prime-boost with BCG, H56:IC31 has delayed and
reduced clinical disease in Mtb-challenged cynomolgus macaques and prevented
reactivation of latent infection (Lin et al. 2012). The vaccine showed high immuno-
genicity in healthy adults with or without previous disease (Luabeya et al. 2015).
H56:IC31 completed a phase I trial at SATVI, South Africa. The vaccine was safe
and tolerated at all tested doses. It was immunogenic in adult pulmonary TB patients,
who have successfully completed TB treatment [NCT02375698]. A phase II study is
underway to evaluate the safety profile of H56:IC31 in HIV-uninfected, remotely
BCG-vaccinated adolescents (NCT03265977).
ID93 + GLA-SE consists of four antigens of Mtb (Rv2608, Rv3619, and Rv3620,
which are associated with virulence, and Rv1813, the latency antigen) in a novel
glucopyranosyl lipid adjuvant-stable emulsion (GLA-SE; TLR4 agonist). This
fusion protein-adjuvant combination induced antigen-specific polyfunctional CD4
Th1 cells (IFN-γ+ TNF-α+ IL-2+) and reduced the number of Mtb in the lungs of
mice and guinea pigs that were infected with virulent or multidrug-resistant Mtb
strains. Further, the vaccine elicited polyfunctional effector CD4 T cell and CD8 T
cell response in BCG-vaccinated or Mtb-exposed human PBMCs (Bertholet et al.
2010). GLA-SE is known to induce polyclonal Th1 response and signals through
both MyD88 and TRIF in vivo (Orr et al. 2013). Synergistic interaction between
MyD88 and TRIF is required for Th1 cell polarization with a synthetic TLR4
agonist. A phase IIa trial for this vaccine was completed in South Africa in
HIV-negative TB patients that have recently finished treatment for pulmonary TB
disease [NCT02465216]. A future phase IIb trial is being planned to investigate the
prevention of recurrence of disease in the same population.
M72 + AS01E is a subunit vaccine, consisting of fusion protein of Mtb antigens
Mtb32A (pepA, probable serine protease pepA, Rv0125) and Mtb39A (Rv1196, a
PPE family protein) in adjuvant AS01E, which enhances cell-mediated immunoge-
nicity. AS01E is an adjuvant in liposome formulation with monophosphoryl lipid A
(MPL) and QS21 immunostimulants. Several phase IIa studies were completed and
phase IIb studies are planned for this vaccine candidate. Safety and immunogenicity
of M72 was tested in different populations, as a booster to BCG in the Gambian
infants, in HIV-infected adults in combination with antiretroviral therapy in the
Switzerland, and in healthy PPD-positive adults in the Philippines (Idoko et al.
2014; Thacher et al. 2014). A randomized phase II study was conducted for safety,
reactogenicity, and immunogenicity of M72 + AS01E in three cohorts: (i) TB-naïve
adults, (ii) adults previously treated for TB, and (iii) adults who have completed the
intensive phase of TB treatment. In addition, phase II trials on safety and immuno-
genicity of M72 + AS01 were also conducted on adults with TB and AIDS patients
[NCT01755598]. The study ended prematurely because of high incidence of red-
ness/swelling reactions at the site of inoculation (Gillard et al. 2016).
VPM1002 is a recombinant BCG vaccine developed at the Max Planck Institute
of Infection Biology (Grode et al. 2013; Nieuwenhuizen et al. 2017). Phase I study
396 G. Kaur et al.
confirmed the safety and efficacy of the vaccine candidate. The vaccine was safe and
well tolerated in healthy volunteers and led to the induction of multifunctional CD4
T cell and CD8 T cell immune response [NCT01113281; NCT00749034]. Phase II
study to evaluate safety and immunogenicity of VPM 1002 in comparison with BCG
in newborn infants in South Africa was found to be as safe and well tolerated as BCG
[NCT01479972]. VPM1002 has teamed up with Serum Institute of India Pvt. Ltd.
(SIIPL). Together they are conducting a phase II trial in South Africa to evaluate
efficacy and safety in HIV-exposed and HIV-unexposed infants. In addition, a phase
III trial is being planned in India to assess the potential of VPM1002 as a postexpo-
sure vaccine in prevention of TB recurrence after successful drug therapy.
RUTI is a polyantigenic liposomal vaccine made of detoxified, fragmented Mtb
cells (FCMtb). It facilitates a balanced Th1/Th2 response to a wide range of antigens.
Standard treatment for latent TB infection (LTBI) requires isoniazid (INH) from 6 to
9 months, which presents important compliance problems. RUTI was developed to
generate a polyantigenic immune response and to reduce the treatment duration of
latent TB (Cardona and Amat 2006). Immunization with RUTI vaccine further
pronounced the decrease in the colony-forming units, as compared to treatment
with rifampicin and isoniazid (Cardona et al. 2005). Treatment with RUTI showed
no signs of toxicity following chemotherapy in mice and guinea pigs. Phase I studies
showed dose-dependent local adverse reactions and elevated the T cell response in
healthy volunteers (Vilaplana et al. 2010). In another study, RUTI was shown to
have potential for both prophylaxis and immunotherapy for TB infections (Vilaplana
et al. 2011). A phase II clinical trial demonstrated reasonable tolerability of RUTI at
three different doses with the induction of local abscess in HIV-infected and
HIV-noninfected individuals (Nell et al. 2014). RUTI induced stronger activation
of IFN-γ-secreting CD4 T cells and CD8 T cells against tuberculin purified protein
derivative, ESAT6 and Ag85B (Guirado et al. 2008). The main target for RUTI is
MDR-TB and it is being implemented in phase IIa study in patients with MDR-TB.
TB/FLU-04L is a mucosal vectored vaccine based on an attenuated replication-
deficient influenza virus vector expressing antigens Ag85A and ESAT-6 and influ-
enza virus strain A/Puetro Rico/8/34 H1N1. It was designed as a prophylactic boost
vaccine for infants, adolescents, and adults. It has completed a phase I study
[NCT02501421] and a phase IIa trial in people with LTBI is being planned for
this vaccine (WHO 2017).
21.13 Vaccines in Phase III Clinical Trials
Some of the vaccines against TB have reached phase III clinical trials. M. vaccae is a
whole-cell-based heat-killed immunotherapeutic vaccine. It consists of heat-
inactivated M. vaccae. It is the only therapeutic vaccine recommended for TB
immunotherapy by the WHO ( 2017; Stanford et al. 2004). It is licensed by the
China Food and Drug Administration as an immunotherapeutic agent to help shorten
the treatment for patients with drug-susceptible TB. A phase III trial to study efficacy
and safety in China found vaccine to be well tolerated with no serious adverse effects
(WHO 2014). Heat-killed M. vaccae when added to chemotherapy of TB patients
showed improved sputum conversion and x-ray appearances. Immunostimulating
effects of M. vaccae were attributed to their cell wall, which elicit more production
of Th1 cytokines. Phase III trial to assess its immunogenicity and safety in
preventing latent TB is completed. It is the largest TB vaccine clinical trial in the
past decade, involving 10,000 people aged 15–65 years. The MIP was safe when
used as a therapeutic vaccine in combination with drugs in sputum-spear positive
pulmonary TB patients (Sharma et al. 2017).
21.14 Summary and Future Prospects
Vaccination is the most effective and economically viable strategy to improve

public health. The use of vaccine has saved millions of lives. Some of the diseases
such as smallpox, polio, and measles have almost been eradicated through vacci-
nation. Unfortunately, this could not be achieved in the case of TB. Tremendous
efforts have been made by scientific community world-wide in understanding the
protective efficacy of vaccines. A reliable vaccine that can eliminate TB globally is
yet far from reality. The appearance of drug-resistant strains of Mtb is a stern
warning to further expand our horizon of understanding molecular mechanism of
how 85–95% of Mtb-infected individuals remains protected throughout their life
and only 5–15% develop active TB. Further, it is quite crucial to deeply evaluate
the sequence of events being generated during host-pathogen interaction. More
importantly, the variable protective efficacy of BCG and its failure in TB-endemic
areas create a more complex situation for the development of universal vaccine.
Therefore, it demands additional knowledge of interfering factors such as NTMs,
LTBI, and helminths that affect the host immunity against pathogens.
Recently gut microbiota, environmental and nutritional factors have been
suggested to contribute substantially in preventing or provoking diseases
(Sathyabama et al. 2014; Khan et al. 2016; Feng et al. 2018). Furthermore, in
TB-endemic population, prior immunity developed due to exposure of NTMs and
LTBI interferes with antigen processing and presentation of BCG. Thereby, the
immune system fails to optimally expoilt BCG antigens to elicit long-lasting
memory T cells. Consequently, a vaccine that does not require extensive antigen
processing and induces enduring memory T cell response would be an ideal
vaccine for TB-endemic world. To gain an in-depth understanding of disease
pathogenesis, a rationalized vaccine should be multi-epitope, composed of pro-
miscuous CD4 T cell and CD8 T cell epitopes from latent, active, chronic, and
drug-resistant forms of Mtb. The vaccine should bear an adjuvanting moiety
through which it can be targeted to APCs, in particular DCs. Based on these
justifications, peptide vaccines may be quite successful in the future against TB
for endemic and non-endemic inhabitants.
398 G. Kaur et al.
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Immunotherapeutic Potential
of Mycobacterium indicus pranii Against 22
Tuberculosis
Sangeeta Bhaskar and Bindu Singh
Abstract
Mycobacterium indicus pranii (MIP), a nonpathogenic saprophytic mycobacte-
rium, is potential tuberculosis (TB) vaccine candidate as it shares a large number
of cross-reactive antigens with M.tb and has demonstrated unique ability for
immunomodulation. Studies in animal models of TB have demonstrated its
significant prophylactic and therapeutic efficacy against TB. The immunological
aspects of MIP-mediated protection include induction of Th1 and Th17 type of
response, activation of macrophage effector functions, and CD4+ T cell activation
along with enhanced CD8+ T cell cytotoxic activity, thereby resulting in inhibition
of the intracellular growth and multiplication of M.tb. When used as a booster to
BCG vaccine, MIP conferred higher protection in animal models of
TB. Protective efficacy of killed MIP vaccine has been established in clinical
trials. Immunotherapeutic potential of this vaccine is confirmed in a recently
conducted multicentric clinical trial in category II TB patients, having advanced
disease, and was difficult to treat. Moreover, practical implementation of MIP as
TB vaccine has a major advantage as it is a cost-effective vaccination strategy for
effective control of TB with no adverse effects.
Keywords
MIP · Tuberculosis · Vaccine
S. Bhaskar (*) · B. Singh

Product Development Cell-I, National Institute of Immunology, New Delhi, Delhi, India
e-mail: sangeeta@nii.ac.in

https://doi.org/10.1007/978-981-32-9413-4_22
408 S. Bhaskar and B. Singh
List of Abbreviations
MIP Mycobacterium indicus pranii

MW Mycobacterium leprae
Mw Mycobacteria
MAC Mycobacterium avium complex
PLC Phospholipase
BCG Mycobacterium bovis
RIF Rifampicin
INH Isoniazid
PYZ Pyrazinamide
ETB Ethambutol
ATT Antitubercular treatment
PTB Pulmonary TB
22.1 Mycobacterium indicus pranii
There are more than 150 recognized species in the genus “Mycobacterium.” Many of
these species are pathogenic that poses serious health threats to humans such as
M. tuberculosis (causing tuberculosis) and M. leprae (leprosy) while others are
saprophytic. In the late 1970s, Talwar et al. identified Mycobacterium w (Mw) from
a group of 16 strains of atypical mycobacteria which gave highest immune response
against M. leprae. The nomenclature “Mw” initiated as an outcome of the coding
given to those atypical strains of mycobacteria investigated for the development of
vaccine against leprosy, where mycobacteria coded as “w” was found to be
promising. At that time, no overlap was found with the name Mw and names of
other known mycobacterial species, but later on Mycobacterium w (Mw) was
renamed as Mycobacterium indicus pranii (MIP) as there was confusion with
hyper-virulent M. tuberculosis-W strain of Beijing genotype family. This name
was given on the basis of location of its isolation from India (indicus), discovered
by Prof. Pran Talwar (pranii), and characterization at the National Institute of
Immunology (pranii).
In 1980s, immunotherapeutic activity of MIP was evaluated by clinical trials in
multi-bacillary leprosy patients along with chemotherapy. Immunotherapy with MIP
once every 3 months combined with chemotherapy significantly enhanced bacillary
clearance and shortened the recovery time (Zaheer et al. 1993). It was found to
be effective in multi-bacillary leprosy patients with high bacteriological index
(Talwar et al. 1990; Talwar 1999). Besides therapeutic benefits, it showed
immunoprophylactic efficacy in household contacts of leprosy patients (Sharma
et al. 2005). Killed MIP vaccine subsequently got approval of the Drug Controller
General of India and is now manufactured and marketed by an Indian pharmaceuti-
cal company under the name “Immuvac.”
22 Immunotherapeutic Potential of Mycobacterium indicus pranii Against Tuberculosis 409
Later on molecular phylogenetic analysis and genome-wide comparisons

revealed that MIP is a predecessor of Mycobacterium avium complex (MAC)
which comprises of opportunistic pathogens. Comprehensive analysis of cellular
and biochemical features together with chemotaxonomic markers and when com-
parison with other mycobacterial species establishes specific attributes of MIP
(Ahmed et al. 2007; Saini et al. 2012). Analysis of the study suggested that MIP
can be placed at a transitory position, in between saprophytes and pathogens. This
unique placement has given it the advantage of sharing a large number of antigens
with M.tb as compared to other nonpathogenic vaccine candidates. MIP has moder-
ate growth rate (time of colony appearance 6–8 days) that is in between slow-
growing (3 weeks for M.tb) and fast-growing (3 days for M. smegmatis) mycobacte-
rial species. Whole-genome sequence analyses provided evidence of substantive and
interesting differences between MIP and M.tb as well as other members of the
M. tuberculosis complex. Comparison of “antigenicity index” of MIP with M.tb
antigens, based on the analyses by VaxiJen, suggested that MIP possess unique
ability for immunomodulation (Saini et al. 2012).
MIP has shown immunomodulatory activity in various diseases (Ahmad et al.
2011; Katoch et al. 2008; Rakshit et al. 2012; Talwar 1999). Killed MIP vaccine is
approved for human use against leprosy. Further in animal studies, MIP was found to
be protective against TB and human clinical trials as immunotherapeutic/
immunoprophylactic candidate vaccine are underway (Gupta et al. 2009, 2012a, b;
Saini et al. 2012). MIP has broad-spectrum immunomodulatory potential as it shares
a large number of B and T cell antigens with other mycobacteria, viz., M.tb and
M. leprae (Saini et al. 2012; Yadava et al. 1991).
22.1.1 Genomic and Proteomic Characteristics
Whole-genome sequencing of MIP could explain the attributes, responsible for its
unique lifestyle and also molecular basis of immunomodulation. Its genome size is
approximately 5,589,007 base pair and has mosaic architecture. The genome
consists of 5270 ORFs. The mean G + C content of MIP genome measures up to
68%. Approximately, 34% genes of MIP are laterally acquired and these acquisitions
are found to be crucial to MIP physiology. It has 66 genes from PE/PPE family
(50 PPE and 16 PE), out of which five genes are unique to MIP (Saini et al. 2009,
2012). Crucial role of PE/PPE family in host immunomodulation was highlighted by
antigenic analyses between M.tb and MIP. One hundred fifty-two members of
PE/PPE multigene family exhibit antigenicity index > 0.4, out of which 141 proteins
have amino acid sequence similarity with MIP proteins.
MIP proteome was elucidated by BLAST analysis. Till now, 80% of MIP
proteins have been annotated. Out of these, 7.5% proteins were found to be unique
in MIP with no remarkable homology with proteins present in M.tb proteomes.
Analysis of protein coding genes revealed that about 82% of M.tb proteins have
homology with MIP proteins (Rahman et al. 2014; Singh et al. 2014).
22.1.2 Nonpathogenic Characteristics of MIP
Various attributes of MIP were revealed by annotation of genome which explained

nonpathogenic nature of MIP. The presence of PE-PPE genes and mce1 operon
enables MIP to invade the host cells, but it does not have mce2 and mce3 operons
which are required for persistent macrophage infection, whereas pathogenic
mycobacteria M. avium and M.tb have both mce2 and mce3 operons which enable
their survival inside the macrophages. MIP also lacks phospholipase (plc) ABCD
genes, which help mycobacteria to use host fatty acids as a potential source of carbon
during persistent infections. The devS/devR two-component system, essential for
survival in low oxygen conditions, is also absent in MIP. MIP also lacks gene trpD,
which is important for tryptophan biosynthesis. Due to the absence of these genes,
MIP cannot survive in the host for prolonged periods of time and thus, it is not able
to cause latent infection. On the other hand, MIP possesses abundant catalases and
superoxide dismutases, which help in mitigating oxidative stress. Complete cyanide
and thiocyanate biodegradation machinery is present in MIP which is responsible for
degradation of thiocyanate to produce CO2 and NH3, which in turn are used by MIP
as a source of carbon and nitrogen for limited survival intracellularly. Ten ORFs of
MIP genome having significant similarity to hemerythrin genes could be responsible
for the survival of MIP for a short period in the anoxic conditions.
These nonpathogenic attributes of MIP were confirmed by in vivo experiments in
different animal models which established that infection with MIP is self-limiting
and clears off within 4–6 weeks, which probably makes an ideal scenario. A whole
bacterial vaccine which can persist for a restricted duration in the host to induce
enough memory T cells but also in due course of time removed by the immune
response of the host without causing any adverse pathology is an important desired
feature of a vaccine for its efficacy as well as from safety point of view.
22.2 Protective Efficacy of MIP Against Tuberculosis
As tuberculosis continues to be a serious health problem, there are intensive efforts

to find new vaccines to control this disease. The present vaccine against tuberculosis
(TB), Mycobacterium bovis (BCG), has poor protection against the pulmonary TB in
adults. It is general perception that active TB occurs when immune system is
weakened, which otherwise keeps M.tb in check when fully competent. This
suggests that natural immunity fails to control the pathogen for long time in subset
of individuals and is not sufficient. Whole bacterial vaccines are one of the important
vaccine strategies. This approach has advantage of multiple antigens and built-in
adjuvant activity. Mycobacterial strains sharing antigens with M. tuberculosis are

being evaluated as alternatives to M. bovis for vaccine use. Being whole organisms,
these vaccines are able to induce broad-spectrum immune response (both humoral
and cellular immune responses) as compared to subunit vaccines. Following this
approach, MIP has been studied for its immunomodulatory potential against TB and
leprosy.
Earlier studies have reported that MIP gives protection against tuberculosis in
both BCG responder and also in non-responder mice strains (Singh et al. 1991).
Protective potential of MIP and the underlying immune responses were further
studied in the animal models of tuberculosis. Generally, live bacteria are known to
give higher protection than killed one as live bacteria persist in the host for some
time which results in strong memory response. Route of immunization also has an
important role. Aerosol inhalation is a non-invasive delivery method that physically
targets the lung for its pharmacological effect and induces both local and systemic
immune response. Protective efficacy of live and killed forms of MIP via parenteral
route and through aerosol immunization was compared with BCG (Gupta et al.
2009). MIP immunization by both the routes gave higher protection than BCG in the
animal models of tuberculosis. Live MIP vaccine by aerosol route gave significantly
higher protection as compared to BCG. More importantly, MIP immunization also
provided significantly higher long-term protection as compared to BCG against TB.
MIP induces Th1 and Th17 type of response whether given in killed or live form.
It activates macrophage effector functions as well as lymphocytes activity. Besides
activation of CD4+ T cells and IFN-γ secretion, MIP also induces significant CD8+ T
cell cytotoxic activity. Activated macrophages are able to restrict the intracellular
growth and multiplication of M.tb (Gupta et al. 2009). Protective efficacy and
modulation of immune response post-M.tb infection was further evaluated in sus-
ceptible guinea pig model. Pathogenesis in guinea pig model resembles human
tuberculosis. At different postinfection time points, reduction in bacterial load,
improvement in lung pathology, and organized granulomatous response were
observed in the MIP-vaccinated group as compared to BCG-immunized group
(Gupta et al. 2012b). In MIP-immunized group, higher protective Th1 response
was observed as compared to BCG group at early time point after M.tb infection and
moderate Th1 response at late chronic stage of infection. Inside the granuloma of
infected lungs higher activation of antigen-presenting cells was observed in the
MIP-immunized group.
22.2.1 MIP Immunotherapy as an Adjunct to Chemotherapy

for Treatment of TB
The current chemotherapy [the multidrug regimen comprising of rifampicin (RIF),

isoniazid (INH), pyrazinamide (PYZ), and ethambutol (ETB)] available for TB
usually results in poor compliance as these drugs are to be taken for a minimum of
6–9 months. Although, new cases of TB can be cured by currently available drugs
and to improve the compliance, WHO has introduced the “directly observed ther-
apy” but still patients who default on therapy develop severe risk of relapse and
acquiring drug resistance. To effectively eliminate the replicating and persistent
bacteria from the infected lungs, multipronged strategy is needed.
During the course of tuberculosis infection, the protective Th1 immune response
is suppressed and immunosuppressive Th2 response gets elevated which facilitates
the survival of M.tb and progression of disease. Immunotherapy strategy which
could boost the Th1 immune response in infected person might prove to be effective
as it can act synergistically to chemotherapy. Efficacy of MIP as an adjunct to
standard chemotherapy for TB was evaluated in animal models, when given by
aerosol or subcutaneous route. MIP immunotherapy by intranasal route resulted in
increased infiltration of antigen-specific lymphocytes and macrophages in the M.tb-
infected lungs which resulted in killing of bacteria in synergistic way along with
chemotherapy. MIP treatment along with standard drugs resulted in further reduction
in bacterial loads and less pulmonary pathology as compared to “only drugs” treated
group (Gupta et al. 2012a). A balanced inflammatory and suppressive immune
response was observed in the late stage of “drug+MIP immunotherapy” treatment,
which helped in the control of initial inflammatory reaction and restoration of normal
lung tissue. Recent study suggested that autophagy induced by MIP plays an
important role in reversal of phagosome maturation block in M.tb-infected
macrophages, which leads to accelerated clearance of M.tb when MIP immunother-
apy is given by nasal route (Singh et al. 2017). Combined results of different studies
suggested that MIP could be potentially very useful in eliminating the persistent
bacteria when given as an adjunct to standard chemotherapy. For induction of early
local immune response in the lung, MIP immunotherapy by intranasal route can play
a very crucial role.
22.2.2 MIP as a Booster to BCG Vaccine Confers Higher Protection

in Animal Models
BCG, the current vaccine against TB, protects against childhood tuberculosis but is
poorly effective in adults. Since BCG has given significant protection against
pediatric TB in endemic areas, prime-boost strategy is the most practical approach
for control of TB. MIP shares a large number of antigens with M.tb and BCG and
also had been shown to give higher protection as compared to BCG in animal models
of TB. With this background, efficacy of MIP was evaluated as a booster to BCG
vaccine in animal models of tuberculosis. MIP booster enhanced the BCG-mediated
immune response and resulted in higher protection as evident from the reduction in
bacterial load in the lungs of infected animals of BCG-MIP group as compared to
only BCG-immunized group (Fig. 22.1). Evaluation of pulmonary pathology
Fig. 22.1 Bacterial loads at 4 and 8 weeks after M.tb challenge. Bacterial load in different groups
was evaluated in lungs after 4 weeks (a) and 8 weeks (b) of M.tb challenge. Significantly reduced
colony-forming units (CFU) per gm of tissue were found in “BCG-MIP” regimen as compared to
“only BCG”-vaccinated group. Data represents the mean CFU SD of six guinea pigs in each
group [P 0.05; P 0.01; P 0.001]
demonstrated a reduced number of macroscopic lesions, cavities, and hemorrhagic

spots in BCG-MIP group as compared to unvaccinated and BCG only groups.
Aerosol route of MIP booster immunization proves to be more effective in protection
than subcutaneous route (Saqib et al. 2016). It was observed that Th1 and Th17
cytokines were upregulated in infected lungs of “BCG-MIP” group in contrast to
“only BCG” group. Higher levels of multifunctional T cells with high MFI for
TNF-α and IFN-γ were observed in the BCG-MIP group after M.tb infection.
Findings of the study demonstrated that MIP can be given as a booster to BCG
vaccine for efficient protection against TB, which might be a very cost-efficient
approach for effective control of TB.
22.3 Clinical Trials
In 1990s, prophylactic efficacy of MIP against leprosy was evaluated in a population

of about 30,000 people from 272 villages in Kanpur (India). The population was
vaccinated with two doses of MIP at six-month interval. Healthy contacts of leprosy
patients who had no evidence of TB disease were given MIP vaccine/placebo. After
follow-up for 13 years, retrospective analysis of the data showed that incidence of
tuberculosis was reduced significantly in the MIP-vaccinated group as compared to
placebo group in that randomized placebo-controlled study (Katoch et al. 2008).
Further higher protection of MIP was observed in the subgroup, vaccinated with
BCG in the childhood suggesting that booster dose of MIP could enhance the
protective efficacy of BCG. That observation formed the basis of detailed animal
studies where MIP was evaluated as booster to BCG vaccine.
Later, in one of the recently conducted clinical trials, MIP was given as an adjunct
to chemotherapy in Cat II pulmonary TB patients. This was double-blind, placebo-
controlled, multicentric trial where MIP demonstrated significantly higher cure rate
in difficult to treat cases, i.e., those with high bacillary load, drug resistance, and/or
with bilateral cavities. At 4 week after the start of therapy, sputum culture conversion
in a significantly higher number of patients (67.1%) was observed in the MIP group
compared to the placebo (57%) group (p ¼ 0.0002). Early sputum conversion in the
MIP group suggested that it played an important role in clearance of M.tb. This has
important implications in controlling the spread of M.tb as live bacteria in the sputum
are major contributors for sustained incidence of tuberculosis. MIP has come a long
way. Recently a phase III, double-blind, placebo-controlled clinical study has started
to evaluate its efficacy in preventing TB in healthy household contacts of pulmonary
TB patients.
22.4 Safety of MIP Immunization
Toxicity studies were done with heat-killed MIP in mice as well as guinea pigs and
no untoward reactions were observed in these experimental animals. Killed MIP
vaccine was found to be safe and well tolerated in clinical trials. Safety profile of live
MIP was also studied and no detectable MIP was observed in any organs after
30 days of immunization, provided by any route. No pathological symptoms were
observed in various organs at any time point after immunization. A normal lung
parenchyma was retained, the interalveolar septae were thin, and no cellular
exudates were observed in the alveolar spaces at any time point post-aerosol
immunization (Gupta et al. 2012a, b). Infection with live MIP in mice and guinea
pigs is self-limiting, which is crucial for the induction of robust memory response. A
desired feature of a vaccine is that it should persist long enough for generation
of memory T-cells but still should be gradually removed by the host immune
response without developing any adverse pathology. MIP, being a nonpathogenic
bacterial vaccine with exceptional properties of immunomodulation, has an advan-
tage for the practical application of this vaccine to masses and could be very cost-
effective strategy for effective control of TB (Fig. 22.2).
Fig. 22.2 Pulmonary pathology at 4 weeks after infection. Shown are the representative images of
lungs (a) and spleen (b) of guinea pigs from different groups at 4 weeks postinfection. The presence
of pathological lesions like nodules, hemorrhagic spots, and cavities in the lungs was macroscop-
ically examined. Spleen size was also measured and compared among the groups. (c) Pictures
showing H&E-stained whole lung sections. (d) H&E-stained sections visualized at 40 X magnifi-
cation. (e) Gross pathological score and (f) percent healthy alveolar tissue in infected lungs. Data
represents the mean SD [P 0.05; P 0.01; P 0.001; comparisons were made with
“only BCG”-immunized group]
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TB Diagnostics: Journey from Smear
Microscopy to Whole Genome Sequencing 23
Himanshu Vashistha and K. K. Chopra
Abstract
Tuberculosis is not just a basic clinical and general issue of the whole world. It
has attracted worldwide attention of the entire scientific majority because of its
alarming rate of incidence and mortality. Since history of early examination of
tuberculosis and its drug resistance, it has been proved that the diagnosis
upgrades that enhances the survival and early case detection related to early
treatment initiation. Careful bacteriological assurance and general prosperity
approach to manage TB rely upon initial microscopic examination, solid & liquid
culture methods, rapid molecular and standard DST assays.
For each assay of detection, there are new procedures and advancements
adopted since their inception. Developing diagnostic techniques having access
to quick accurate results, near-patient point-of-care diagnostics are now vital to
curb and control TB and HIV-TB burden in resource-poor countries. This chapter
has focused on the journey of those diagnostic techniques which remain the
mainstay of TB diagnosis from early diagnostic work of smear microscopy to
latest techniques involving whole genome sequencing. Starting late it is basic to
comprehend that at present, there is no autonomous test for the quick acknowl-
edgment of tuberculosis in all patients. While some new frameworks are direct,
others have complex components and require sophisticated infrastructure and
trained manpower. It is therefore imperative to choose from different diagnostic
assays individually or to combine them inside a country’s national TB control
program.
H. Vashistha
Viral Hepatitis Division, National Centre for Disease Control, New Delhi, Delhi, India
K. K. Chopra (*)
New Delhi Tuberculosis Center, New Delhi, Delhi, India

https://doi.org/10.1007/978-981-32-9413-4_23
420 H. Vashistha and K. K. Chopra
Keywords
Tuberculosis · Diagnostics · Culture · Next-generation sequencing
Abbreviations
AFB Acid-fast bacilli

ATT Anti-tubercular therapy
CBNAAT Cartridge-based nucleic acid amplification test
CDC Center for disease control
CFP Culture filtrate protein
CFU Colony-forming unit
CRI Colorimetric redox indicator
DB Database
DR Direct repeat
DST Drug susceptibility testing
ESAT Early secreted antigenic target
FIND Foundation for Innovative Newer Diagnostics
FM Fluorescent microscopy
IFNg Interferon gamma
IGRA Interferon-gamma release assays
INH Isoniazid
IS6110 Insertion sequence
IVD In vitro diagnostics
LAM Lipoarabinomannan
LAMP Loop-mediated amplification PCR
LED Light-emitting diode
LJ Lowenstein Jensen
LM Liquid medium
LPA Line probe assay
LTBI Latent tuberculosis infection
MDR Multidrug resistance
MIRU-VNTR Mycobacterium interspersed repeated units-variable number of
tandem repeats
MODS Microscopic observation of drug susceptibility
MTBC Mycobacterium tuberculosis complex
NGS Next-generation sequencing
NRA Nitrate reductase assay
NTM Nontuberculous Mycobacterium
23 TB Diagnostics: Journey from Smear Microscopy to Whole Genome Sequencing 421
OADC Oleic acid, albumin, dextrose, catalase

PANTA Polymyxin B, azlocillin, nalidixic acid, trimethoprim,
amphotericin B
PPV Positive predictive value
RFLP Restriction fragment length polymorphism
RIF Rifampicin
RNTCP Revised National Tuberculosis Control Program
rRNA Ribosomal ribonucleic acid
SLID Second-line injectable drugs
SPR Surface plasmon resonance
TAT Turnaround time
TB Tuberculosis
TTD Time to detection
VOC’s Volatile organic compounds
WGS Whole genome sequencing
XDR Extensive drug resistance
ZN Ziehl Neelsen
23.1 Background
Tuberculosis (TB) has been a torment of the mankind, an ancient scourge. Histori-
cally, the consummate immensity inflicted by TB is matchless with other diseases on
the human race in terms of morbidity and mortality. Consequences of TB have
momentous outcome on human existence especially in terms of social and economic
part. In history, TB has ruled over in terms of killing millions of people till date;
however other diseases like smallpox and plague have also killed many people, but
their reign has been relatively short-lived. TB has changed its face as the time has
lapsed and evolved itself to dominate over the entire cure. However, even today TB
remains as a daunting enemy frightening to exterminate humans.
Despite many advances in the twentieth century, tuberculosis still executes
somewhere in the range of 2–3 million individuals worldwide every year and is
rejuvenated in many parts of our world (Drobniewski et al. 2003). Mycobacterium
tuberculosis is the source of infection which is responsible for tierce of the earth’s
human population (nearly 2 billion people) (Drobniewski et al. 2003). The current
pandemic is alarming due to the publishing of multiple drug resistance and increas-
ing comorbidities. In this chapter, we discuss about the most accepted diagnostic
techniques innovated in laboratory diagnosis of the opportunistic pathogen and a
devastating killer known as “the Mycobacterium tuberculosis”.
23.2 Epidemiology
TB is among the top ten leading causes of mortality worldwide especially from
solitary infectious agent. Approximately, 10.4 million people fell ill with TB in
2016: adults counted for 90%, males 65%, people with HIV (74% in Africa) 10%,
and five countries contributing to 56%, India, Indonesia, China, the Philippines, and
Pakistan (WHO Global TB report 2017).
In 2016 alone, 6.3 million new instances of TB were accounted for (up from 6.1
million in 2015), proportionate to 61% of the evaluated occurrence of 10.4 million.
In 2016, 41% of the 3.4 million new bacteriologically affirmed and recently treated
TB cases told internationally were reported to have used rifampicin, up from 31% in
2015 (WHO Global TB Report. 2017). Immediate and exact determination of TB,
HIV-related TB, and drug-resistant TB, trailed by arrangement of treatment in
accordance with global norms, anticipates well-being among individuals who
build up the infection. Expanding access to diagnosis ahead of schedule and precise
determination utilizing a WHO-prescribed fast demonstrative is one of the three
principle destinations of TB lab fortifying endeavors under the End TB Strategy
(WHO Global TB Report 2017).
23.3 Diagnosis
Diagnosis reveals a disease or condition by laboratory-based investigations. To

comprehend why presently our world is impairing to end TB, we need to understand
tuberculosis (TB) diagnosis. Treating a TB-infected patient involves finding and
diagnosing the cause first. In the field of TB, challenge starts at the very first step,
i.e., diagnosing the case. TB is identified by detecting Mycobacterium tuberculosis
in a patient clinical specimen. Prompt confirmation of tuberculosis is still a demand-
ing problem in paucibacillary and extrapulmonary forms.
Traditional TB diagnosis banks on medical history, tuberculin skin test, chest
X-rays, smear microscopy, and bacteriological examination. For survival, rapid
determination of tuberculosis and its susceptibility toward drugs is a requisite and
by screening infectious cases fosters contact tracing, enactment of institutional cross-
infection plan of action, and other public health operations.
Therefore, the urgent need for rapid diagnosis and confirmed recognition of active
M. tuberculosis disease that can be achieved at the point of care, i.e., outside the
traditional laboratory setting, is still in quest even after several diagnostic modalities.
The fresh dynamism of copious policy makers, funding agencies, and grants
promotes the need to foster innovations that deliver better diagnostic tools having
higher confidence while using affordable approaches. Following detection, treatment
and complete eradication of the TB menace and its associated risks can be taken with
confidence to fulfill End TB Strategy goals.
23.3.1 Laboratory Diagnosis of Tuberculosis
Tuberculosis history is equally matured to human history. Hints of it have been

found from Neolithic entombment locales somewhere in the range of 7000 years of
age and in antiquated Egyptian mummies. Hippocrates distinguished the ailment,
calling it “phthisis” or “consumption,” and thought that it was lethal. Anyway until
the seventeenth century because of progressively exact neurotic and anatomical
illustration of the illness being recorded, TB started to be distinguished. Early
detection work: During 1720, Benjamin Marten (an English clinician) recommended
that consumption, purported in light of the fact that it appeared to devour individuals
from inside, could be brought about by “wonderfully minute living creatures.” It
would at long last be left to German doctor Robert Koch to really “see” them by
utilization of a recoloring procedure called staining. Prior work on isolation of the
tubercle bacilli by the bacteriologist was first introduced in Berlin on 24 March 1882.
Utilizing exceptionally arduous techniques, Koch could disengage Mycobacterium
tuberculosis by utilizing Tindall’s blood serum medium (Brock 1999).
TB Laboratory Diagnostic Tool Development: The Objectives

The general objective for enhancing TB diagnostics is to help the advancement of
economical understanding of patient-focused applications on basic innovation
stages, fitting to various levels of developing and underdeveloped nation well-
being frameworks. This should prompt generally speaking enhanced access to
proper medicines and/or treatments.
Objectives for TB Diagnostic Test Development
• Ease the process of TB case detection, including smear-negative, extrapulmonary,

and pediatric TB, by raising the sensitivity and specificity and expanding
improved approachability
• Development of simple, accurate, safe, rapid, and inexpensive assays that are
targeted for point-of-care level usage in the healthcare system and reducing the
turnaround time (TAT) to few hours
• To authorize more efficacious TB treatment monitoring
• To screen drug resistance rapidly in both first- and second-line anti-TB drugs
• To dependably recognize latent TB infection and decide the danger of movement
to dynamic (active form) infection, empowering the reasonable utilization of
preventative treatment
23.4 See the Bug
23.4.1 Smear Microscopy
Microscopy stood as a mainstay of tuberculosis control since TB diagnosis initiation,

due to its ability to capture sputum-smear-positive (most infectious) cases, is less
time consuming and costs less, although found to be low in specificity. Sputum
smear microscopy is developed more than a century ago; the presence and/or
absence of acid-fast bacilli is determined in a sputum sample using a microscope
in this technique.
It is for the most part hard to analyze TB when the bacterial load is under 10,000
for each milliliter of the sputum test, giving wrong negative outcomes for a few
patients (Mudur 2017). From this time forward, smear microscopy affectability is
expanded by utilizing different fluorochrome dyes, for example, auramine and
rhodamine, and fluorescent-recoloring procedures. Not only fluorescence micros-
copy sensitivity is 10% higher than conventional Ziehl-Neelsen (ZN) microscopy,
but also the examination of fluorochrome-stained smears takes less time. Major
shortcomings in fluorescence microscopy are its high cost, due to expensive mercury
vapor light sources, periodic maintenance, and need of a dark room. The combina-
tion of light-emitting diodes (LED) and fluorescent light output for fluorescent
microscope (FM) has developed a simple yet robust LED FM microscope, which
requires less power along with no dark room requirement. The endorsement of WHO
has established LED FM as an alternative to light microscopes (LMs) especially in
resource-limited settings (Minion et al. 2011).
Smear microscopy detects most infectious pulmonary tuberculosis cases. The
quality of smear microscopy is crucial in TB diagnostics in resource-limited settings
as the performance is dependent on its execution (Rieder et al. 2007; CDC
Homepage [https://stacks.cdc.gov/view/cdc/31282] 2000).
Low sensitivity (25–75% in respect of culture) and more bacilli requirement for
positivity (approx. 5 103–104 bacilli per ml) are the major limitations. Sensitivity
and the positive predictive value (PPV) are affected by scores of aspects (CDC
Homepage [https://stacks.cdc.gov/view/cdc/31282] 2000; WHO Roadmap 2010;
Stop TB Partnership 2008), namely, prevalence and infectiousness of the disease,
the type, consistency and quality of the sample provided, mycobacteria number in
the sample and smear preparation quality, and staining and observing process.
Mycobacterial species are not identified and also the bacilli viability is not detected
in the sample through smear microscopy. In case of HIV-TB-coinfected patients and
patients with paucibacillary samples, smear microscopy result is mostly negative and
requires diligent screening to identify scanty AFB bacilli (Figs. 23.1 and 23.2).
23.4.1.1 Histopathology
Laboratory diagnosis of extrapulmonary tuberculosis is done through histopath-
ological examination of tissue specimens. The sensitivity mainly depends on ease
of sampling and site-specific sample aspiration. Reduced granuloma formation in
patients with HIV infection hampers the specificity. Trained experts and resource
facilities for histopathology are commonly unavailable in many low-resource coun-
try settings.
Advantages Sputum smear microscopy is an easy, inexpensive, and rapid method

in quickly detecting infectious cases of pulmonary TB, having sufficient number of
AFBs captured easily by sputum microscopy.
Fig. 23.1 Fluorescent

microscopy result from a
direct sputum sample in a (3+)
AFB-positive TB patient
Fig. 23.2 ZN smear

microscopy of M. tuberculosis
culture positive, showing
characteristic cord formation
Disadvantages Smear microscopy requires at least 5000 bacilli per milliliter of

sample for a positive result in direct microscopy. In patients suffering from
extrapulmonary TB, along with HIV coinfection and also those with nontuberculous
mycobacteria (NTM) infection, it is documented that sensitivity of smear micros-
copy is further reduced.
Limitations NTM are not differentiated from MTBC, while dead and viable bacilli
are also not distinguished. Moreover, drug-resistant forms are not differentiated from
susceptible strains.
23.4.2 Grow the Bug
23.4.2.1 Culture
Sputum mycobacterial culture served as the “gold standard” diagnostic assay in TB
diagnosis, and it also facilitates DST for definitive diagnosis of drug-resistant forms
Fig. 23.3 Characteristic colony morphology of M. tuberculosis

(a) Solid culture on LJ medium. (b) Image showing rough, buff, tough, ivory-colored MTB colony
morphology. (c) Liquid culture MGIT-positive tube showing flaky growth
of tuberculosis. Because of the higher sensitivity of culture, capturing of only few

numbers of bacilli (approximately 10 bacilli/ml in contrast to a minimum of 5000
bacilli/ml of sputum for microscopy) is also possible. Detection of a number of TB
cases is increased by 30–50% with the use of culture techniques. Moreover, for
species identification and drug susceptibility testing (DST), cultures are more useful
(Migliori et al. 2012; De Kantor et al. 1998).
One of the major touchstones for accurate recognition of Mycobacterium tuber-
culosis is culture, but more time consumption and often no recovery in
paucibacillary specimens are significant constraints. However, requirement of dedi-
cated laboratory with technical expertise for mycobacterial culture and DST are
often not routinely available in high-burden countries with TB, limiting their useful-
ness. The turnaround time for conventional sputum mycobacterial culture is 6–8
weeks and therefore the definitive diagnosis is delayed.
Solid Media
Egg-based media: Lowenstein-Jensen (LJ) medium is an egg-based enrichment
media, having malachite green (inhibitor of non-mycobacterial organism), espe-
cially for sputum culture. Glycerol in LJ favors M. tuberculosis growth, while LJ
lacking glycerol instead consisting sodium pyruvate benefits M. bovis growth
(Leao et al. 2004) (Fig. 23.3a, b).
Ogawa medium, an LJ lacking asparagine, nonselective, egg-based medium, is
refrigerated for long-term usage even after few months, if tube caps are tightly closed
to minimize evaporation.
A drawback is that if contamination occurs, it engulfs the whole surface of the

slant, resulting in culture loss. For specimens with less bacillary load, it may take
three to eight weeks for culture to be positive.
Agar-based media: Most commonly used, Middlebrook 7H10 and 7H11 media
are often formulated in the laboratory from powdered agar bases, by adding
Middlebrook oleic acid-albumin-dextrose-catalase (OADC) enrichment. Typical
cord formation is a characteristic feature of M. tuberculosis and can be counted as
early as one week after incubation because of the clearness in 7H10 and 7H11 plates.
In addition, colonial morphology on agar plates is better visible as compared to
egg-containing slants, which is helpful for the identification of mycobacteria. Slant
tubes and/or plates are prepared and are less likely to become contaminated.
Middlebrook 7H11 has little advantage over 7H10 as it contains 0.1% casein
hydrolysate, which helps in recovery of isoniazid-resistant mycobacteria. In addi-
tion, 7H11 supports better growth of multidrug-resistant (MDR) strains, lacking
7H10 agar plates (Iseman 2000).
Liquid Media
Liquid media support rapid growth than solid media. TAT for Middlebrook 7H9
liquid medium takes 7–14 days, Middlebrook 7H11 agar takes 18–28 days, and LJ
medium takes 21–42 days (Mase et al. 2007).
For the fulfillment of the aim of early growth of M. tuberculosis, numerous
diagnostic methods and modifications have been developed during the last four
decades. In the 1980s, semiautomated and automated liquid culture systems became
available, which facilitated rapid growth and detection of M. tuberculosis with a
TAT of about 10 days (Palomino 2012).
Few essential methods, adopted by RNTCP in India and also endorsed through
WHO, are the following:
(i) BACTEC system: This assay depends on utilization of substrate palmitic acid
converting to radioactive carbon dioxide. In this framework, a colorimetric
sensor recognizes the creation of CO2 broke down in the culture medium. This
assay is based on generation of radioactive carbon dioxide. In this system, a
colorimetric sensor detects the production of CO2 dissolved in the culture
medium. TAT is 5–10 days.
(ii) MGIT (mycobacteria growth indicator tube): MGIT tube contains modified
Middlebrook 7H9 broth, OADC growth supplement, and mixture of antimicro-
bial compounds known as PANTA (polymyxin B, amphotericin B, nalidixic
acid, trimethoprim, azlocillin). Growth is detected, based on O2 consumption
by aerobic microorganisms, through a detection system (nonradioactive in
nature) which utilizes fluorochromes for detection and drug screening. Early
growth detection (7–12 days) is done and is useful for rapid phenotypic drug
susceptibility (Fig. 23.3c).
(iii) ESP culture system II: In this method, M. tuberculosis growth detection occurs
due to estimation of rate of oxygen consumption which is present within the
upper headspace of the culture vials.
Advantages Identification of M. tuberculosis from culture confirms TB diagnosis,

which significantly increases (30–50%) the number of cases. Due to its high
sensitivity, it can detect cases even in smear-negative cases. Culture not only
provides the necessary isolates but also serves as a base for putting up the
conventional DST.
Disadvantages Culture medium are highly prone to contamination and expensive.

It requires dedicated facilities and infrastructure for media preparation, specimen
processing, and growth of organisms, along with skilled laboratory personnel and
specialized biosafety conditions.
Limitations Specimen decontamination is necessary prior to be inoculated in

culture to prevent overgrowth by other microorganisms. Even culture is not 100%
sensitive, as all decontamination methods are also harmful to mycobacteria. Culture
is less sensitive in specimens with paucibacillary load. Although gene amplification
methods are nowadays much more sensitive, they pose chances of false positivity
and reason being primarily contamination (Rodrigues et al. 2009).
23.5 Identify the Bug
23.5.1 Identification of Mycobacterium tuberculosis Complex
Since biochemical tests are time taking in identifying M. tuberculosis complex, in

2007, WHO recommended liquid culture assays, DST, and rapid speciation (strip
speciation) tests based on the detection of a TB-specific antigen from positive
cultures, for confirmation of M. tuberculosis complex (Capilia TB; Tauns
Laboratories Inc., Shizuoka, Japan (Shen et al. 2009) helping in rapid diagnosis of
TB and MDR-TB).
23.5.1.1 Chromatographic Immunoassay

A qualitative identification method for differentiating Mycobacterium tuberculosis
complex from positive cultures. Certain diagnosis of TB actually occurs when
M. tuberculosis complex is differentiated from other organisms in a clinical sample
which is culture positive in solid or liquid media. As M. tuberculosis complex strains
(except some sub-strains of M. bovis BCG and nontuberculous mycobacteria)
predominantly secrete the MPB64 protein, diagnostic discrimination can be done
between M. tuberculosis complex and nontuberculous mycobacteria. The principle
reaction using monoclonal antibodies against MPB64 has been developed and
evaluated as the basis of such assays (Abe et al. 1999; Hillemann et al. 2005; Hirano
et al. 2004).
Lateral Flow Assays Available BD (Becton, Dickinson and Company) Diagnostic

Systems, Sparks, MD, USA; SD Bioline MPT64 assay, SD Bioline assay, Capilia
TB-neo, TAUNS Laboratories Co, Numazu, Japan; and BD MGIT TBc identifica-
tion test (Fig. 23.4a, b).
Fig. 23.4 Lateral flow assays showing positive bands for confirmation of MTB complex
(a) Positive LJ culture with positive M. tuberculosis complex result in SD Bioline MPT64 assay
(b) Positive M. tuberculosis complex result in BD TBcID assay
23.5.1.2 Serological Methods

Serological methods use blood serum and can also be easily applied in resource-
limited settings, therefore being an attractive choice as a diagnostic tool. In contrast,
false positivity remains a major limitation in antibody detection with less defined
preparations in high-burden countries like India.
Screening of people having latent TB infection requires the development of new
diagnostic tools and standards to curb the scourge (Thillai et al. 2014; Diel et al.
2012). Interferon-gamma release assays (IGRAs) were developed to diagnose LTBI;
however tuberculin skin test (TST) proved to be the least expensive test (Thillai et al.
2014). The TST and IGRA work on the principle of capturing the interaction of T
cells to TB antigens (Cruz-Knight and Blake-Gumbs 2013).
Tuberculin Skin Testing (TST)

MTB protein, namely, tuberculin, a derivative mix of proteins (PPD), is injected into
a person in subcutaneous tissue intradermally. With previous exposure to mycobac-
terial proteins in the vaccine or having any latent MTB infection, a skin reaction
occurs due to type IV delayed hypersensitivity as an immunologic response (Thillai
et al. 2014). Skin induration reaction size is measured with readings at 48 and 72 h
and a standard cutoff value of 10 mm. However, false-positive responses are
documented in persons who are BCG vaccinated previously during infancy and
false-negative responses also documented in immunosuppressed individuals. In
India, TST is being used in diagnostic algorithm for pediatric age group as a
supportive evidence.
IGRAs
Interferon-gamma release assay test was developed, as the percentage of false
reporting is high in interpretation of tuberculin skin test. Two available examples
of IGRA assays are Quantiferon-TB Gold and T-Spot TB test. The IGRA assay
diagnosis detects MTB antigens, including the early secretory antigen target
6 (ESAT-6) and culture filtrate protein 10 (CFP-10), both which stimulate
interferon-gamma production in the host.
The IGRAs are more sensitive (81%) and specific (88%) which is 10% more than
70% sensitivity noted in TST (Diel et al. 2012). Detection of cytokine IFN-g is made
which is released from T cells, reacting antigens not present in the BCG vaccine
(Diel et al. 2012; Goldman and Schafer 2011). The release of IFN-g is measured in
the blood sample from the suspected patient.
Immunodiagnostic tests are not universally accepted due to their cross reactivity
and poor sensitivity especially in high-TB-burden countries (with exception of
tuberculin skin test). Indian government has banned the use and also the sale of
these diagnostic tests because of their high false reporting issues.
Transformation of latent infection to active TB form in healthy individuals and its
overall understanding overlay foundations in better future diagnostics. Identifying
risk factors involved in high and/or low-TB-burden settings will definitely solve the
equation of the complex immune response in TB, and its better understanding will
lead to the invention of more useful diagnostic tools which can change today’s
scenario.
23.5.1.3 Drug Resistance Detection Using Phenotypic Assays

Rapid reporting of drug susceptibility testing to first-line drugs and/or second-line
drugs requires detection of drug resistance and is the key for the earlier implementa-
tion of treatment in a bacteriologically confirmed case. Phenotypic drug susceptibil-
ity testing (DST) methods are performed in solid or liquid media as direct or indirect
tests.
Direct testing involves a batch of drug-containing and a drug-free media, on
which a concentrated specimen is inoculated directly. In contrast, in indirect testing,
a pure culture cultivated from the original specimen is inoculated on the drug-
containing media.
Phenotypic methods such as indirect testing assays are extensively validated and
are honored as the gold standard. Three methods are commonly used: proportion,
absolute concentration, and resistance ratio. Moreover, DST results among the
three methods for first-line anti-TB drugs usually did not vary.
DST methods are based on growth in solid media using a manual system and
using both automated and manual systems in case of liquid medium. For predicting
the results, direct observation and calculation of growth by colony count method in
solid media and in liquid broth media were made through oxygen consumption due
to CO2 production or enzymatic modification of the media.
First-line DST Drug sensitivity testing techniques forecast result to one medica-
tion tested as a delegate from a group of medications (e.g., rifampicin results cover
rifampicin and rifapentine, prothionamide covers ethionamide, and the other way
around). First-line anti-TB drugs incorporate isoniazid (H), rifampicin (R), etham-
butol (E), and streptomycin (S), despite the fact that streptomycin is currently not
part of most standard medications, and pyrazinamide (Z), which is normally part of
the standard treatment yet, isn't viewed as a first-line testing drug because of the
trouble in getting dependable outcomes. Rifampicin and isoniazid results are repro-
ducible most accurately than streptomycin, ethambutol, and pyrazinamide results.
After confirmation of MDR-TB, rapid susceptibility testing of additional first- and
second-line drugs needs to be performed following current WHO recommendations
(WHO Policy Statement 2008).
Second-Line DST Assay It is a challenging, multiplex, and high-priced method.

Well-promoted and endorsed liquid culture and DST methods and the standardized
solid proportion method are validated. Automated liquid systems are recommended
for second line drugs, as the current gold standard phenotypic DST method.
MDR-TB cases are often treated by more potent second-line group of anti-TB
drugs, e.g., the injectables (kanamycin, capreomycin, and amikacin), the
fluoroquinolones (ofloxacin, levofloxacin, and moxifloxacin), ethionamide or
prothionamide, clofazimine, cycloserine, and linezolid. Aminoglycosides, cyclic
peptides, and fluoroquinolones have already proved their reliability and reproduc-
ibility, promising a quality assured diagnosis of XDR-TB.
After almost four and a half decades, news regarding novel drugs for MDR/XDR-
TB treatment have raised some considerable hope. These drugs have been developed
following several trials and they have also succeeded in receiving provisional
approval. Two examples are bedaquiline, a drug from diarylquinolone family
(developed by Janssen Pharmaceutica), and delamanid, a dihydro-
nitroimidazooxazole derivative; both are also being used in MDR TB treatment in
some countries after approval. Regulatory approval for use of bedaquiline and
delamanid in MGIT DST method is still in trial phase in high-burden countries.
DST Using Noncommercial Methods These DST methods are less expensive and
having lack of standardization as it is vulnerable to errors and demographic
modifications in adopting the methodology. Hands-on expertise is mandatory in
performing these methods following good laboratory practices (GLP), best
microbiological techniques, and with more appropriate quality assurance, backed
up with enough training. WHO has selectively reviewed the performance for avail-
able noncommercial culture and DST methods that were providing adequate results
under controlled laboratory protocols in reference/national laboratories (WHO Pol-

icy Statement 2010a, b).
The most common methods incorporate microscopic observation of drug suscep-
tibility (MODS), colorimetric redox indicator (CRI) methods, and the nitrate reduc-
tase assay (NRA).
MODS Specimens inoculated to drug-free and drug-containing liquid culture

media are compared. Following this, early growth is observed in microcolony
method through microscopic examination. Hence this method is also recommended
for rapid screening of patients suspected with MDR-TB.
CRI Methods Liquid culture medium having a specific colored indicator in a

microtiter plate is used and reduced after in vitro exposure of M. tuberculosis strains
to anti-TB drugs. Moreover, this method has got several recommendations as an
indirect test on M. tuberculosis isolates from MDR-TB suspects. Time to detection
(TTD) of MDR-TB is not quicker than conventional DST methods using commer-
cial liquid culture or molecular line probe assays but certainly cheaper.
NRA The ability of M. tuberculosis to reduce nitrates forms a basis of detection

through a colored reaction in this assay. It is therefore recommended as a direct
and/or indirect method on solid culture, for screening of MDR-TB suspects. It
acknowledges that time to detection of MDR-TB is comparably slower than con-
ventional DST methods using solid culture.
23.6 Multiply the Bug’s DNA
23.6.1 Drug Resistance Detection Using Genotypic Assays
The use of molecular biology is imperative in the rapid diagnosis of mycobacteria.

Methods based on capturing nucleic acids have taken the place of most of the
classical methods as a rapid screening tool. These assays adaptability is advanta-
geous primarily, as directly used on the sample material, or culture, and even can be
used as a rapid and unequivocal species differentiation assay from culture material
has made them successful screening platforms. Molecular genetic tests offer signifi-
cant time advancements in the identification of mycobacteria, thereby permitting
quick initiation of resistance tests and specific treatment. They are useful tools for the
detection and differentiation of mycobacteria from cultures and can have a high
specificity and sensitivity. Also, they must be placed in the reinforcement of the
conventional diagnostic workup, and therefore, assay results should always correlate
by standard methods also.
PCR-Based Assays
Biochemical tests are mostly used for identification of mycobacteria for a long time
now (traditional practice), but due to longer TAT, the results are ambiguous; hence,
PCR-based assays emerged and were implemented later on. PCR is specific for gene
amplification assays, needs careful interpretation, and therefore must be in strong
correlation with other parameters such as clinical, histological, cytological, biochem-
ical, imaging, and therapeutic specifications while making final conclusion, espe-
cially in drug-resistant TB diagnosis.
Line probe assays (LPA)

LPA have been developed and implemented in most of the countries in their
National Tuberculosis Control Program worldwide after endorsement from WHO.
LPA, a PCR-based multiplex genotypic DST assay, fulfills the objectives of screen-
ing of rifampicin and isoniazid drug resistance and simultaneously identifies
M. tuberculosis complex in the patient sample directly.
(a) INNO-LiPA: INNO-LiPA (RIF & MYCOBACTERIA v2) are two different line
probe assay kits used for the drug resistance detection and identification of the
genus Mycobacterium and 16 distinct mycobacterial species, respectively.
Abiding to the manufacturer’s interpretation criteria, the following Mycobacte-
rium species can be differentiated simultaneously: M. tuberculosis complex,
M. kansasii, M. xenopi, M. gordonae, M. genavense, M. simiae, M. marinum,
M. ulcerans, M. celatum, MAIS, M. avium, M. intracellulare, M. scrofulaceum,
M. malmoense, M. haemophilum, M. chelonae complex, M. fortuitum complex,
and M. smegmatis. This kit performs on the basis of nucleotide differences in the
16S–23S rRNA spacer region and can be done on either liquid or solid cultures
(Scarparo et al. 2001; Tortoli et al. 2003). The INNO-LiPA test comprises three
major steps: DNA extraction, DNA amplification, and hybridization.
(b) Genotype line probe assay: The GenoType series is based on the patented DNA
strip technology and it permits the genus Mycobacterium to be genetically
differentiated to different species.
The Hain CM test kit is designed to identify the most common mycobacteria, and
another AS assay differentiates additional Mycobacterium species further. Species
differentiation among the M. tuberculosis complex is done by GenoType MTBC.
Only after the culture turns out to be positive, the CM, AS, and MTBC strips can be
able to detect and analyze mycobacteria but not from patient material directly.
The GenoType MTBDRplus V2 test simultaneously detects M. tuberculosis com-
plex and its inherent drug resistance to rifampicin and/or isoniazid by screening most
common mutations in the rpoB and katG/inhA (high/low isoniazid resistance) genes,
respectively (Nikolayevskyy et al. 2009) (Fig. 23.5a).
Fig. 23.5 In figure (a), DNA

strip is interpreting MDR
results in an MTBDRplus V
2.0 kit
In figure (b), DNA strip is
showing interpretation of
MTBDRsl results of an
XDRTB patient
The GenoType MTBDRsl V2 detects not only the confirmation of M. tuberculosis

complex but also its resistance to second-line anti-TB drug groups, namely,
fluoroquinolones by screening mutations in the gyrA and gyrB genes, and/or
second-line injectable drugs (SLID), aminoglycosides/cyclic peptides by mutations
in the rrs gene (injectable antibiotics as capreomycin, viomycin/kanamycin,
amikacin), and/or low-level kanamycin resistance by mutations in the eis gene
(MTBDRsl kit insert 2018) (Fig. 23.5b).
Although endorsed by WHO and other international recommendation agencies,
these tests have some limitations. A major shortcoming of the DNA probe technol-
ogy is its incapacity in identifying mixed cultures. However, additional probes might
yield a result in case of any mixed cultures. Additionally, a little number of species
only are differentiated by these additional probes. Moreover, these MTBDRplus and
MTBDRsl assays are not validated on smear-negative specimens and
extrapulmonary specimens, confirming to the kit limitations. Vanquishing such
limitations, specific gene probes coupled with hybridization, polymerase chain
reaction-based assays, and DNA sequencing platforms have been adopted
nowadays.
(c) Cartridge-based nucleic acid amplification test (CBNAAT): Majority of real-

time PCR assays have now been implemented in rapid and specific detection of
M. tuberculosis in the clinical specimens. CBNAAT assays have high sensitivity

unlike smear microscopy particularly in HIV-positive people and less time-
consuming ability (less than two hours) comparing to sputum culture (several
weeks). Along with the rapid detection of drug resistance, it shows the unique-
ness and easy implementation of this technology.
The Xpert MTB/RIF from Cepheid A quantum leap in TB diagnostics is inven-

tion of a nucleic acid amplification test that is fully automated, as sputum smear
microscopy turns out to be a century-old technique. Xpert after endorsement from
WHO has been rolled out in many countries including India for the rapid screening
of rifampicin drug resistance and also adopted by technical and operational
guidelines in India for tuberculosis control program. Xpert MTB/RIF is also
recommended as a stand-alone diagnostic screening test in individuals at risk of
MDR-TB. The use of GeneXpert has not only diminished the TTD for rifampicin
drug resistance significantly from days to only two hours, but also the near-patient
device can be operated with minimal laboratory infrastructure requirement in a
mobile van to reach outskirt areas. However, GeneXpert does not obliterate the
need of conventional microscopy, culture, and anti-tuberculosis drug sensitivity that
are required to monitor the progression of treatment and for drug resistance determi-
nation to drugs other than rifampicin (ISTC 3rd edition 2014). Recent developments
from Cepheid show promise and commitment to develop and implement other ATT
drugs cartridges, which are used in MDRTB treatment including second-line drugs
also (Fig. 23.6a, b).
Fig. 23.6 Above figure (a) showing GeneXpert system with a cartridge and figure (b) showing
GeneXpert result in a patient sensitive to rifampicin
(d) TrueNat: The TrueNat TB test (in-house product from India) has shown
promising results as a new molecular test that can diagnose TB in one hour
and simultaneously detect drug resistance to rifampicin. The TrueNat machine is
designed more likely as a point-of-care machine, is semiautomated, and can
handle outdoor situations without electricity and fulfills the objective with
maximum conclusion on minimum prerequisites. Sample testing can be done
as soon as a suspected TB patient with symptoms is seen. The system is battery
operated, and with the help of a handheld device, it can be used in far-flung areas
in the periphery (Chaitali et al. 2014). The procedure involves DNA extraction
which spends 25 minutes initially and another 35 min to diagnose TB, which is
an additional one hour for testing of rifampicin drug resistance (The Hindu
Webpage 2017).
(e) Loop-mediated isothermal amplification (LAMP): TB LAMP is a manual
nucleic acid amplification test comprising of DNA amplification along with
gene detection in a single step. This test can be used at outreach centers as a
near-patient assay and does not require sophisticated instruments and laboratory
infrastructure (manufactured by Eiken Chemical Company Ltd (Tokyo, Japan)
(Eiken Chemicals Webpage 2017)).
Advantages LAMP is rapid with turnaround time (TAT) of 40 min; it gives result
usually observed through the naked eye under ultraviolet light. Additional 40% more
patients are detected due to increased sensitivity than smear microscopy as in smear-
negative samples where bacillary load is less. Once smear microscopy results are
known and documented, it can also be implemented as an add-on test (WHO Policy
Guidance 2016).
Limitations It does not differentiate among drug-resistant TB and drug-sensitive

TB, and it is having less specificity as compared to smear microscopy, thereby
producing increment in false-positive results.
(f) Genedrive: Recently TB detection and rifampin-resistant TB detection are now

possible through a new assay based on NAAT platform. Genedrive Company
(formerly called Epistem) developed Genedrive, but its low accuracy is not
supporting the WHO requirements.
Limitation Sensitivity was found to be zero percent in smear-negative samples and

specificity 45.6% in smear-positive samples which is considered suboptimal com-
pared to smear microscopy. More number of false-negative result, failing to detect
many cases, provides no use over smear microscopy. Failing TB detection even in
direct smear-negative culture-positive samples, resulting in poor assay performance
(Shenai et al. 2015).
By and large, quality enhancement techniques for gene amplification are built up
to be exceedingly sensitive and explicit for analysis of tuberculosis (specificity)
straightforwardly from clinical examples. Contingent on the bacteriological status
and copy number of target sequence, affectability (sensitivity) has extended from
70% to 100% though explicitness (specificity) between 80% and 100% has been
accounted for different modalities.
23.7 Typing the Bug
23.7.1 Molecular Typing of Mycobacterium tuberculosis
Molecular typing involves DNA fingerprinting of M. tuberculosis strains, thereby

improving the understanding in complexity of TB transmission. Moreover, the
recognition of genotype families has facilitated many research groups working on
the population structure of the M. tuberculosis complex and exploring its transmis-
sion dynamics. Several molecular typing assays for Mycobacterium tuberculosis are
now available, with disparate levels of reproducibility, discriminative power, and
technical competence requirement.
Spoligotyping (Spacer Oligonucleotide Typing)

Spoligotyping is a direct, less expensive, quick, and reproducible assay to contem-
plate the phylogeny of M. tuberculosis complex strains and to connect phenotypic
highlights of disengages with the genotype family the Mycobacterium speak
to. Clinical specimens of M. tuberculosis complex are recognized by the presence
or absence of at least one spacer.
Polymorphism in the direct repeat (DR) locus is detected in the mycobacterial
chromosome. Direct repeats conserved regions (36 bp) are interspersed with unique
spacer sequences (from 35 bp to 41 bp) in size. 43 spacer sequences are used in
spoligotyping for M. tuberculosis complex strains. Multiple copies of variable DNA
spacers in the genomic DR region of M. tuberculosis complex isolates are produced
through PCR. Amplified PCR products are further hybridized on a membrane having
covalently bound spacer oligonucleotides, deduced from DR region sequences
(Brudey et al. 2006). The SpolDB4, a publicly available database on the
M. tuberculosis complex, is one of the largest and contains spoligo patterns from
approximately 40,000 clinical isolates representing 122 countries. Spoligotyping is
more commonly applied to identify the genotype family; and this technique is less fit
for strain typing.
MIRU-VNTR
Mycobacterium Interspersed Repetitive Units - Variable Number Tandem Repeats
typing (MIRU-VNTR) assay is developed to identify the genetic polymorphisms
within bacterial species. MIRU-VNTR works on the basis of the number of tandem
repeats differences in the 24 stretches of the genome of M. tuberculosis complex
strains. Using amplification, up to 24 loci are amplified with the help of primers
specific for the flanking regions of each repeat locus; later on to deduce the number
of tandem repeats present, sizes of the amplified stretches of tandem repeats are
determined (Kremer et al. 2005). A numerical code is generated by the detection of
number of tandem repeats at the different loci. That code serves as a DNA fingerprint
of the respective tuberculosis bacteria. VNTR typing has multiple applications, most
commonly contact tracing, source-case finding, and ruling out transmission reliably.
Advantage Its results are easy to compare and is user-friendly than IS6110 RFLP
typing. It is mostly suitable as a high-discrimination typing method for highly
conserved genotypes.
RFLP Typing
Restriction fragment length polymorphism (RFLP typing) assay detects the number
of IS6110 insertion sequences present in the genome difference in strains (Kremer
et al. 1999). Due to the presence of highly variable genomic insertion sites in
M. tuberculosis complex strains, highly variable banding patterns can be obtained.
Since it uses its high level of discrimination power and reproducibility, RFLP assay
proves to be the most extensively used method for typing bacterial strains over the
last few years.
Advantage Although requiring highly experienced manpower and sophistication,

RFLP is still superior because of the level of discrimination. Therefore it is also used
for strain typing.
23.8 Sequencing the Bug
23.8.1 DNA Sequencing
DNA sequencing involves the amplification and identification of genetic mutations

in different genomic regions. It also offers a fast and specific identification of
mycobacterial species and drug resistance simultaneously. The principle used in
DNA sequencing methods is the determination of species-specific nucleotide
sequences, its comparison to quality-controlled sequences known widely available
from MTB databases. Although DNA sequencing was invented by F. Sanger in 1977
known as chain termination sequencing, latest developments in the NGS technology
were only made after the complete sequencing of Mycobacterium tuberculosis
genome in 1998 (Cole et al. 1998). There is a rapid surge in development of
diagnostic assays using DNA sequencing platform mostly involving
pyrosequencing, next-generation sequencing (NGS), and/or whole genome sequenc-
ing (WGS).
Pyrosequencing
Pyrosequencing strategy depends on the ongoing real-time sequencing of short
stretches at the time of DNA synthesis (sequencing by synthesis) and has
demonstrated as a promising new instrument for the fast distinguishing proof of
mycobacteria with positive cultures as well as utilizing processed smear-positive
sputum examples. This strategy is less demanding to perform and is more affordable
than conventional sequencing, yet the shorter DNA stretches are not as discriminat-
ing. Automated sequencers can provide results within 1 to 3 days (Pai et al. 2009).
Routinely DNA sequencing is labor-intensive and can cost a lot in countries with
less infrastructure and poor health settings.
Whole Genome Sequencing (WGS)

Whole genome sequencing provides comprehensive data on resistance mutations
and strain typing for monitoring transmission, but unlike for conventional molecular
tests, this has previously been achievable only from cultures of M. tuberculosis.
Detection of known resistance mutations provides a personalized treatment of drug-
resistant tuberculosis as compared to empirical treatment. This personalized therapy
not only helps improved outcomes but also spares the use of resistant antimicrobials
providing a basis of universal DST-guided treatment. Studying whole genomes also
allows simultaneous identification of all known resistance mutations as well as
specific markers with which transmission can be monitored (Brown et al. 2015a, b).
Small fractions of M. tuberculosis genome are interrogated with the help of
classic genotyping methods that target highly variable genetic elements. Hence,
small evolutions occurring in other genomic regions are not captured. However,
NGS assay gives access to nearly full genome sequences. Frequent advancements
and increase in affordability have extended the use of NGS technology for research
and epidemiological studies. Various genetic markers are investigated with the help
of microbial genomics, hampering treatment and infection prognosis. Entire genome
sequencing (WGS) is turning into a moderate and available strategy that can
recognize microevolution inside MTB ancestries as they are transmitted between
hosts (Kwong et al. 2015).
The WGS results in better detection of types of various mutations than other
genotypic assays. Moreover, WGS could avoid false positives occurring due to
polymorphism present within the specific gene regions. Multiple individual DNA
strands can be independently sequenced, providing a true discrimination of
heterogenetic variation (e.g., heteroresistance) and quantitative analysis.
Nowadays, a variety of NGS platforms are available, for example, the most rapid
Oxford Nanopore (minION, PromethION, GridION), Bio-Rad Laboratories
(GnuBio), PacBio (Sequel and RS2), Thermo Fisher (SOLiD, S5, personal genome
machine [PGM], Proton), Qiagen (GeneReader), Vela Diagnostics NGS platform
(Singapore), and the most common Illumina (MiSeq, HiSeq, and NextSeq).
WGS needs culturing of M. tuberculosis, a slow-growing microorganism, for
obtaining affluent amount of genomic DNA, making it a time-consuming assay.
However, recently invented WGS assay can be done without prior specimen culture.
This method is based on specific biotinylated RNA baits which are specific to MTB
and can sequence full MTB genomes directly from non-cultured sputum samples
(Brown et al. 2015a, b). In routine, WGS used for clinical specimens is most
common in high-income countries because of its high cost, dedicated infrastructure,
and requirement of skilled manpower with analysis expertise.
As MTB requires early detection of drug resistance, WGS holds great benefit as a
handshake of diagnostic and epidemiological tool combination in a single assay.
Compared to conventional molecular tests which focus on mutations in hotspot
regions of genes involved in resistance to first- and second-line anti-TB drugs,
data generated from WGS analysis applies holistic approach of creating a genome-
based personalized medicine in healthcare. Sequencing data generated by
investigators and researchers are not yet directly comparable, and universal
databases access is limited for surveillance and studies of epidemiological
importance.
TBDReaMDB is a database available for optimal prediction of drug resistance
mutations; the need of a comprehensive and well-curated database is a must for
better correlating genotypic data with phenotypic drug susceptibility testing data.
These challenges are being addressed by large international consortia (e.g., http://
patho-ngentrace.eu/) in order to accelerate the implementation of NGS-based epide-
miology and diagnostics (Sandgren et al. 2009).
The advent of novel anti-TB drugs or modification in regimens requires align-
ment with novel diagnostic platforms, thereby helping scaled use of new drugs and
in developing appropriate genotypic assays to identify drug-resistant alleles to the
new drugs.
Crude information produced by NGS and WGS is colossal and presents a huge
test to the scientists. Mapping or the genome assembly, base variant calling, and near
phylogenetic investigations are a few NGS information data management
techniques. Aptitude in bioinformatics abilities are required for handling and break-
ing down the information utilizing programming and calculations in algorithms
which are very perplexing and regularly unlinked to one another.
23.9 What We Can Do with the Bug
23.9.1 Future Prospects in TB Diagnostics
Evolution in laboratory diagnosis of Mycobacterium tuberculosis has diminished

time lapsed in identification and drug susceptibility results. Continuous efforts have
been made for increasing reproducibility, improvement of performance, and
minimizing cost and infrastructure requirement. Some of the future projections
that are going to be implemented in TB diagnostics are discussed here:
23.9.1.1 Alere DetermineTM LAM Assay

It is a lateral flow (LF)-based immunodiagnostic strip test for the detection of
lipoarabinomannan (LAM) antigen in urine. LF-LAM support the diagnosis of TB
in HIV-positive adult patients. Patients unable to expectorate sputum, with other
immunocompromised condition, and patients unable to move out will definitely be
benefitted with the use of LF-LAM assay, especially in ill diagnostic geographic
conditions (WHO Policy Update 2015).
23.9.1.2 Cepheid GeneXpert Ultra

An updated version of GeneXpert has already undergone multicentric trials in 2017.
Ultra works on the same principle of CBNAAT as used in the earlier GeneXpert, but
with greater sensitivity is comparatively high in direct smear-negative rather culture-
positive specimens, pediatric specimens, extrapulmonary specimens (notably cere-
brospinal fluid), and especially for paucibacillary specimens mostly from
HIV-positive individuals. CBNAAT Ultra cartridge is renovated with improved
assay performance, resulting in a limit of detection (LOD) of 16 bacterial colony-
forming units (cfu) per ml compared to previously 114 cfu/ml in Xpert® MTB/RIF
(WHO Meeting Report of Technical Expert Consultation 2017).
In addition, XDR TB assay has also incorporated probes for detecting mutations
responsible for resistance toward second-line anti-TB drug groups, namely, INH,
FLQ, and AMG, using asymmetric PCR. Sloppy molecular beacons and fluorescent
melt curve analysis is supporting the PCR phenomenon to identify genotype drug
resistance (Roh et al. 2015). Currently under pilot run, the XDR TB assay release
date and price are not known till date.
23.9.1.3 TRCReady® 80
TRCReady® 80 is a molecular diagnostic tool, developed by Tosoh Bioscience
(Japan) as a stand-alone assay with a capacity of processing up to eight samples, and
requires a computer platform for operation. TRCRapid® M.TB kit is marketed by
Tosoh Company for use on this platform. Fully automated sample purification,
amplification, and detection are incorporated in this assay (Drouillon et al. 2009).
Amplification of MTBC specific target is done by using transcription reverse-
transcription concerted reaction (TRCR) amplification technology. Total fluores-
cence is measured in real time as increase in amplification reactions and takes only
30 minutes to complete.
23.9.1.4 Volatile Organic Compounds (VOCs)

The VOCs here are mycobacterial metabolites derived from the host’s digestion,
incorporating microorganisms in the gut or the host’s reaction to the mycobacterial
disease. Companies have used various names for already developed VOC devices
for, e.g., gas chromatography (Menssana Research Inc., USA), metal-oxide sensors
(Nanosynth Materials and Sensors, USA, and The eNose Company, Netherlands),
and metabolite detection by chemical reaction (Metabolomx, USA). The develop-
ment pipeline still focuses on products that are at varying trial stages (Bruins et al.
2013; Lim et al. 2016).
These TB screening devices are portable and handheld. At the point when a
patient associated with experiencing TB inhales into the device tube, VOCs created
by MTB in the lungs tie with titanium oxide nanotubes immobilized in the gadget.
An electrical impulse is being produced upon that binding and is captured and read
by applications in smartphones. Clinically suspected patients with a positive result
are then recommended to undergo mycobacterial confirmation testing. Some of the
proposed VOC platforms are Breathscanner™ and Aeonose™.
23.9.1.5 Assays for Diagnosing Latent TB Infection (LTBI)

C-Tb, a novel skin test is built up and named by Statens Serum Institut in Denmark,
for distinguishing LTBI. The test estimates the body’s resistant reaction to two
explicit MTB antigens that are not decoded in the BCG immunization: ESAT-6
and CFP10. Improved TSTs assays in pipeline include Generium Pharmaceutical
(the Diaskintest, Russian Federation), and china-based skin test known as ESAT-6
has already undergone phase II trials in China (Sun et al. 2013).
23.9.1.6 Automated Microscopy Platforms

With the advent of automated imaging, the conventional visual examination for acid-
fast bacilli (AFB) by direct sputum smear microscopy is also advanced. Some
examples are:
• The TBDx system: It is a mechanized computerized automated microscopy

platform that is accessible from Signature Mapping Medical Sciences Inc.,
USA. This stage comprises of a great magnifying lens (higher resolution
power) in the microscope and imaging framework that related to a slide holding
adjustment that can peruse up to 200 arranged smears utilizing fluorescent
microscopy in a solitary run (Ismail et al. 2015).
• Capture-XT™: An MTB cell advancement gadget is in development stage named
as QuantuMDx (UK), to enhance the affectability (sensitivity) of SSM past
current fundamental concentration techniques, for example, sputum sedimenta-
tion. The organization has begun to examine the utilization of an MTB cell
concentration innovation as a stand-alone assay, namely, Capture-XT™. The
gadget will be battery worked and will most likely work for 8 h on a solitary
charge. Pilot testing of the innovation has already initiated.
23.9.1.7 Surface Plasmon Resonance (SPR) Sensing

SPR-based biosensors systems having several ultrasensitive detectors will be
expected in the next 5 years, as a fully integrated point-of-care SPR imaging-based
diagnostic system (FU et al. 2008).
23.9.1.8 Use of Nano-particles

Quantum dots 1–6 nm nanoparticles can be functionalized with capture probe or
antibodies (Resch-Genger et al. 2008) coated with capture probe. Mirkin and
colleagues built up a promising nanotechnology-based stage for multiplex
identification of protein and nucleic acids utilizing a sandwich of standardized

magnetic and bar-code probes tag tests (Cheng et al. 2006).
23.9.1.9 Lab on a Chip

Single molecule detection will become possible by the detection of minute amounts
of genetic materials. Assays capable of detecting even single molecules are now
being developed, e.g., cantilever beams and nanotube paddle resonators (Witkamp
et al. 2008). These new detection frameworks could be pioneered to deliver “lab on
chip” frameworks for the identification of pertinent biomolecules.
23.9.1.10 Future NGS Platforms

Smaller, robust, and user-friendly stages for use in comparatively smaller research
facilities are at present being developed. These include Genalysis® from DNAe,
Gene Electronic Nano-Integrated Ultra-Sensitive (GENIUS) from GenapSys, and
the Genia sequencer from the Genia Corporation (all USA). These innovative
devices are proposed to have little cartridge-based frameworks with incorporated
sample amplification and sequencing at a cost focus of US$ 100 or less; be that as it
may, further subtleties are restricted (Pankhurst et al. 2016).
Barriers and challenges are there always, but present achievements and future
probabilities in diagnostics offer tremendous potential and hope. The amalgamation
of interdisciplinary sciences like molecular biophysics, nanobiotechnology, immu-
nology, biochemistry, genomics, biomedical sciences, and bioinformatics gives
ever-upgrading and revolutionizing scientific and logical research and relocating
diagnostic development from the biological to the molecular level as an indicative
improvement from the natural to the atomic dimension. These advancements prom-
ise of future TB diagnostics that are rapid, accurate, and cost-effective and help
address multiple drug-resistant TB strains in high-burden, low-resource countries.
However, stand-alone diagnostic test currently cannot assure of all three “quick,”
“cheap,” and “easy” demands. The gaps in the diagnostic services must be addressed
urgently for eradicating tuberculosis. Clinical diagnosis must be supported with the
early adherence to anti-TB drug therapy for the benefit of public health in manage-
ment of tuberculosis. Selection bias compromised interpretation of assay results, and
partial selection is also involved in failure of many diagnostic assays which has
shown optimistic results initially. Even after decades of research, focus is still on
finding new biomarkers required for better detection that is desperately needed along
with all available diagnostic methods. With the recent enthusiasm of various
stakeholders, policy makers, funding agencies, and grants, the need is now to foster
innovations that deliver state-of-the-art tools to diagnose TB with confidence using
affordable approaches. High-cost techniques from the developed nations need to be
subsidized to support the high-TB-burden countries with resource-limited settings. It
is to be trusted that ongoing advances will prompt the improvement of novel
demonstrative techniques and diagnostic strategies that are relevant to use in devel-
oping and creating countries, to reduce the morbidity and mortality with effective
intervention most urgently required (Table 23.1).
Table 23.1 Summary of newer prospects in TB diagnostics pipeline at various stages (detailed
table is freely available at https://www.finddx.org/dx-pipeline-status/) and cited with permission
from FIND India
In regulatory stage (
In development In validation CE-IVD, FDA, CFDA, or
In feasibility stage stage (design stage similar regulatory
(prototype but not design locked/pilot (manufacturing approval in domestic
lock) manufacturing) in place) country)
TB flow (Salus Discovery, True array Sensitive LAM Sandwich vessel
LLC) MDRTB (Fujifilm) concentration diagnostic
Biomarker type: antigens (Akonni Biomarker type: product (Hunan-Tech
Biosystems) antigens New Medicine)
Biomarker type: Biomarker type: whole
nucleic acid bug
(RNA, DNA)
MTB antigens POC True array Aeonose for TB TB resistance module
assays (multiple) XDR-TB (The eNose series (autoimmune
Biomarker type: antigens (Akonni Company) diagnostics)
Biosystems) Biomarker type: Biomarker type: nucleic
Biomarker type: metabolite acid (RNA, DNA)
Nucleic acid
(RNA, DNA)
Blood/urine cfDNA POC PoC (Bioneer) ID-FISH assay Accupower TB and MDR
assays (multiple) Biomarker type: (ID-FISH real-time PCR (Bioneer)
Biomarker type: nucleic nucleic acid Technology, Biomarker type: nucleic
acid (RNA, DNA) (RNA, DNA) Inc.) acid (RNA, DNA)
Biomarker type:
nucleic acid
(RNA, DNA)
High-sensitivity TB rapid Xpert XDR INFINITY Tuberculosis drug
diagnostic (Cepheid) MDR-TB resistance array kit
(global good) Biomarker type: (AutoGenomics) (Capital
Biomarker type: antigens nucleic acid Biomarker type: Bio-Technologies)
(RNA, DNA) nucleic acid Biomarker type: nucleic
(RNA, DNA) acid (RNA, DNA)
TB Dx (not defined) Omni (Cepheid) Accupower MTB drug-resistant
(Unima) Biomarker type: XDR-TB real- mutation test kits
Biomarker type: antibody nucleic acid time PCR (QuanDx)
(RNA, DNA) (Bioneer) Biomarker type: nucleic
Biomarker type: acid (RNA, DNA)
nucleic acid
(RNA, DNA)
Erythra TB (Erythra Inc.) Q-POC INNO-LiPA Rif Mycolor TK platform
Biomarker type: antigens TB/MDR TB TB (Salubris)
(QuantuMDx) (Fuji-Rebio Biomarker type: whole
Biomarker type: Europe) bug
nucleic acid Biomarker type:
(RNA, DNA) nucleic acid
(RNA, DNA)
SemanticMD AI for TB TB MultiTest AdvanSure Anyplex assays for
(SemanticMD) (Selfdiagnostics MDR-TB MDR/XDR series
(continued)

Biomarker type: others, Deutschland GenoBlot assay (Seegene, Inc.)
imaging GmbH) (LG Life Biomarker type: nucleic
Biomarker type: Sciences) acid (RNA, DNA)
nucleic acid Biomarker type:
(RNA, DNA) nucleic acid
(RNA, DNA)
ReView-TB (ChironX) Molecular stool PrimeSuite TB VersaTrek (Thermo
Biomarker type: others, (multiple) (Longhorn Fisher)
imaging Biomarker type: Vaccines and Biomarker type: whole
nucleic acid Diagnostics bug
(RNA, DNA) LLC)
Biomarker type:
nucleic acid
(RNA, DNA)
Breath test (Avisa Pharma FluoroType QMAC DST Sensititre MYCOTB
Inc.) MTBXDR Ver (Quanta Matrix, MIC Plate (Thermo
Biomarker type: 1.0 (Hain Inc.) Fisher)
metabolite Lifesciences) Biomarker type: Biomarker type: whole
Biomarker type: whole bug bug
nucleic acid
(RNA, DNA)
NS-POC TB detector LabChip-based VereMTB Molecu Tech REBA
(Nanosynth) rapid POCT detection MDR/XDR assays series
Biomarker type: (MicoBiomed) (Veredus (YD Diagnostics)
metabolite Biomarker type: Laboratories Pte Biomarker type: nucleic
nucleic acid Ltd.) acid (RNA, DNA)
(RNA, DNA) Biomarker type:
nucleic acid
(RNA, DNA)
TB Breathalyser (Rapid UltraFast MeltPro MTB
Biosensor System) LabChip real- (MDR-TB, XDR-TB
Biomarker type: antigens time PCR-MDR- kits) (Zeesan Biotech)
TB kit Biomarker type: nucleic
(MicoBiomed) acid (RNA, DNA)
Biomarker type:
nucleic acid
(RNA, DNA)
NA-NOSE (Breathtec RTT TB Lophius LAM-ELISA (Otsuka
Biomedical, Inc.) Biosciences Pharmaceuticals
Biomarker type: antigens Biomarker type: Co. Ltd.)
NA Biomarker type: antigens
Blood host marker POC OMNIgene SPUTUM
tests (Multiple) (DNA Genotek, Inc.)
Biomarker type: protein Biomarker type: NA
(continued)

CYCLE MDR-TB (FRIZ PrimeStore MTM
Biochem GmBH) (Longhorn Vaccines and
Biomarker type: nucleic Diagnostics LLC)
acid (RNA, DNA) Biomarker type: NA
GeneDrive MTB FluoroType MTB VER
(Genedrive PLC) 1.0 (Hain Lifesciences)
Biomarker type: nucleic Biomarker type: nucleic
acid (RNA, DNA) acid (RNA, DNA)
InSilixa HYDRA-1K
(InSilixa, Inc.)
Biomarker type: nucleic
acid (RNA, DNA)
Scanogen (Scanogen Inc.)
acid (RNA, DNA)
MtB Drug Resistance Dx
(Omniome, Inc.)
acid (RNA, DNA)
Low-cost easy-to-use
NGS (multiple)
acid (RNA, DNA)
BioNanoPore
(NanoLogix, Inc.)
Biomarker type: whole
bug
mfloDx MDR/XDR-TB
(EMPE Diagnostics)
acid (RNA, DNA)
Not defined (Mobidiag)
acid (RNA, DNA)
Incipient TB assay
(Abbott Laboratories)
Biomarker type: NA
T-Cell Immune Prof
(Becton-Dickinson)
Biomarker type: NA
QFT-Predict (Qiagen)
Biomarker type: NA
QIA-TB Signature
(Qiagen)
Biomarker type: NA
(continued)

Not defined (Biomerieux/
Bioaster)
acid (RNA, DNA)
Molecular bacterial load
assay (MBLA) (LifeArc/
University of St Andrews
Infection Group)
acid (RNA, DNA)
Acknowledgment Foundation for Innovative Newer Diagnostics (FIND) India deserves a special
acknowledgment for providing permission to cite a table (annexure) summarizing the TB diagnostic
pipeline showing progress at different development stages.
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Breaking the Transmission of TB: A
Roadmap to Bridge the Gaps in Controlling 24
TB in Endemic Settings
Pooja Singh, Jasmine Samal, Sheeba Zarin, Ravikrishnan Elangovan,

Seyed E. Hasnain, and Nasreen Z. Ehtesham
Abstract
Tuberculosis (TB), caused by Mycobacterium tuberculosis (M.tb), remains an
enormous health burden with nearly 2 billion people worldwide being affected by
it, though only 10% progress to active disease. Currently around 58% of people
infected with M.tb are being diagnosed and treated. The lack of a standard research
setting and limited resource in terms of early diagnosis of TB lead to high incidence
of transmission of TB infection in high-TB-burden countries. Therefore, early
diagnosis of TB and an effective vaccination can primarily break the cycle of
transmission of TB. Recommended diagnostic tests, currently available, have
several limitations, making them unsuitable for resource-limited settings and remote
areas. The healthcare settings are the highest risk zones for transmission of drug-
resistant M.tb strains. Multidrug-resistant (MDR) TB is resilient to diagnosis and
pose major complications to patient’s health during treatment. In order to limit the
Pooja Singh, Jasmine Samal and Sheeba Zarin contributed equally with all other contributors.
P. Singh
J. Samal · S. Zarin · N. Z. Ehtesham (*)
R. Elangovan
Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology-Delhi,
S. E. Hasnain

https://doi.org/10.1007/978-981-32-9413-4_24
452 P. Singh et al.
spread of MDR TB, we need to implement better diagnostic tools and health
measures which may eventually interrupt transmission of M.tb from TB patients
to uninfected individuals. Complete and correct execution of regime of anti-TB
therapies at both the individual and community level could help in minimizing the
transmission of TB. This chapter gives an insight on strategies that aid in interrup-
tion of transmission of TB, especially in high-TB-burden areas across the globe.
Keywords
Tuberculosis · Transmission · Mycobacterium tuberculosis · Diagnosis · Vaccine
Abbreviations

GoI Government of India
LPA Line probe assays
LAMP Loop-mediated isothermal amplification
MDR Multidrug resistant
NSP National Strategic Plan
NHP Nonhuman primate
NAATs Nucleic acid amplification tests
RNTCP Revised National Tuberculosis Control Program
RIF Rifampin
TTD Time to detection
TB Tuberculosis
XDR Extensively drug resistant
24.1 Introduction
Tuberculosis continues to be one of the leading causes of deaths in spite of being

declared as a global emergency by World Health Organization (WHO), way back in
1993. The year 2017 recorded 10 million people getting infected with Mycobacte-
rium tuberculosis (M.tb) and the death toll due to TB rose to 1.6 million (WHO
2018). The present rate of decline in TB cases is recorded as 2% per year; however in
order to achieve global END TB target, it is imperative to achieve a decline in TB
cases to as much as 5% annually. An increase in development of drug resistance in
various M.tb strains poses a serious public health threat. In spite of efforts to create
awareness for TB eradication and huge investment in terms of providing access to
primary healthcare and anti-TB drugs, transmission of M.tb remains nearly uninter-
rupted. It is therefore necessary that we reformulate our strategies for tackling TB
cases and derive novel approaches in terms of deciphering novel drug targets,
24 Breaking the Transmission of TB: A Roadmap to Bridge the Gaps in. . . 453
designing new drugs, and generating vaccines. A better understanding of the ways of
transmission and preventing the dissemination of disease from the source can reduce
the overall incidence of TB dramatically (Nardell 2004).
It is estimated that almost three million TB cases worldwide remain undiagnosed
annually (WHO 2014). These undiagnosed cases serve as source for transmission of
M.tb from TB patients to uninfected individuals in the community. TB control
program strategies can be effective in strict sense only if the true burden of TB is
considered, taking into account the underreporting of TB cases. Underreporting of
TB cases could be due to multiple reasons like inaccessibility to TB services,
financial instability of patients, use of obsolete techniques that fail to detect M.tb
in smears, noncompliance of patients to follow up treatment, and poor data recording
(Tollefson et al. 2016). From the patient point of view, a delay or error in diagnosing
TB diagnosis at an early stage of infection not only results in loss of critical time for
timely management of TB but also poses risk in terms of inadvertent transmission of
M.tb to patient contacts. As a result of delay in treatment of TB, patients acquire full-
blown symptoms of the disease and may or may not respond to medicaments, with
greater chances of acquiring multidrug resistance that may increase the duration of
infectivity and may also lead to death. In 2015, only 40% of multidrug-resistant TB
(MDR-TB) cases were estimated out of total 340,000 notified TB cases, globally
(WHO 2017). India poses 27% of the global burden of TB, of which 2.5% are new
TB cases and 16% previously treated TB cases (WHO 2017).
The goal of the National Strategic Plan (NSP) 2017–2025 by the Government of
India (GoI) is to achieve a rapid decline in TB morbidity and mortality that may pave
way for elimination of TB by 2025. The requirements for moving toward TB
elimination have been integrated into four strategic pillars of “Detect-Treat-Pre-
vent-Build” (RNTCP 2017). India’s TB control program to break the TB transmis-
sion cycle has come up with enhanced interventions and strategies such as contact
screening of family members, preventive treatment of all children (<5 years) who
have not been diagnosed with TB, and mandatory compliance to undertake the drug
susceptibility test. To achieve END TB target, there should be universal access to
diagnostic facilities for early identification of presumptive TB cases at the first point-
of-care site, private or public sectors, and prompt diagnosis using diagnostic tests
with sensitivity greater than 90% (Fig. 24.1).
24.2 Approaches to Interrupt the Transmission of TB
(1) Effective TB Vaccination
Vaccines are one of the most effective public health interventions in improving
immunity against infectious pathogens. An effective vaccine is needed to interrupt
transmission of M.tb from TB patients to uninfected individuals, thereby leading to
elimination of TB. Currently, the only vaccine available against TB is the Myco-
bacterium bovis-derived Bacillus Calmette-Guerin (BCG), discovered in 1921
(Calmette 1927). While BCG has shown to protect against severe forms of TB
such as TB meningitis and miliary TB, in infants and children (Rodrigues et al.
454 P. Singh et al.
Early Diagnosis
Case Ways to
finding/Contact break TB Vaccination
tracing transmission
Environmental
measures/health
care settings
Fig. 24.1 A schematic overview of ways to break TB transmission: One of the four essential and
inter-dependable steps for controlling the transmission of TB is to have a vaccination that exhibits
long-term memory response. Early detection of TB is important not only to implement medicament
at an early stage of infection but also in segregating patients from the uninfected individuals.
Healthcare settings can provide easy and correct access to TB treatment regime to the patients.
Preventive healthcare steps such as use of face masks by healthy contacts may prevent inadvertent
acquisition of M.tb from aerosol generated during coughing or sneezing by the patients
1993; Trunz B Bourdin, Fine PEM 2006), however its protective efficacy wanes in
10–20 years. BCG also exhibits limited protection against adult pulmonary TB
(Fine 1995). Such variations in efficacy have been attributed to several factors such
as interaction with environmental mycobacteria, genetic difference of immunized
populations and their immune response to various BCG sub-strains, and geographic
diversity in clinical isolates of M.tb along with some subsidiary factors (Andersen
and Doherty 2005; Kernodle S. Douglas 2010; Springett and Sutherland 1994;
Wilson et al. 1995).
Current vaccination strategies against TB aim to intervene in transmission of M.tb
at three stages: (a) pre-exposure vaccines, aim at prevention of getting infected;
(b) postexposure vaccines, aim at elimination/containment of latent TB and preven-
tion of reactivation; and (c) therapeutic vaccines, aim as an adjunct to chemotherapy.
Several ongoing efforts to achieve promising vaccine candidates are aimed at
improving the immunogenicity of the current BCG vaccine by generating recombi-
nant BCG or by replacing BCG with new platforms such as whole cell lysate or viral
vectors. Table 24.1 summarizes the list of vaccine candidates that are being tested in
various stages of clinical trials. Recently, vaccine candidate M72/AS01 (subunit
vaccine containing mycobacterial antigens 32A and 39A with adjuvant AS01) has
been successful in preventing active TB disease, with an efficacy of 54% in phase 2b
Table 24.1 TB vaccine candidates currently in clinical trials

Preclinical Phase 1 Phase 2a Phase 2b Phase 3
BCG-ZMPI MTBVAC M72 + VPM1002
ASO1
H64:CFA01 Ad5 Ag85A TB/ DAR-901 MIP
Flu04L
PPE15-85A GamTBVac ID93/ H56:IC31 M. vaccae
GLA-SE
CMV6Ag H56:IC31 RUTI
CysVac2/Ad ID93/GLA-SE H4:IC31
BCG-ZMP1 ChadOx1.85A MVA 85A
Aerosol
MVA multiphasic vac
BCG, ChadOx/MVA
PPE15-85A
clinical trial with participants from Kenya, South Africa, and Zambia (Van Der
Meeren et al. 2018). Another vaccine candidate VPM1002, a recombinant BCG
vaccine in which the BCG urease C gene has been replaced with a gene encoding for
listeriolysin from Listeria monocytogenes, is undergoing phase 3 clinical trial in
India (Nieuwenhuizen et al. 2017). In order to check the efficacy of this vaccine in
reducing the rate of relapse and resurgence of TB, this vaccine candidate is being
tested in patients with relapse TB cases. The use of a whole cell bacteria, known as
Mycobacterium indicus pranii (MIP) as an adjunct with the first-line antimicrobial
treatment for Category II TB patients, has shown promising results in the phase 2b
trial and is currently being evaluated in phase 3 trial (Sharma et al. 2017).
The major focus now should be to have an increasing number of vaccine
candidates in the pipeline and to move the current ones from one clinical phase to
another at a faster rate, since vaccine candidate such as MVA85A, in spite of
showing encouraging results (in phase 2b trial), failed to demonstrate additional
protection in infants as compared to BCG (Tameris et al. 2013). There are several
parameters which need to be understood deeply while designing and screening
different vaccine candidates. Firstly, we need to better understand the molecular
pathogenesis and immunogenicity of mycobacterial antigens at the laboratory level
before advancing to clinical stages. Secondly, a systematic and efficient screening of
vaccines in animal models is needed to assess their efficacy in providing protection
and sustained memory response, thereby preventing progression to active disease.
We need multiple assays to be performed using in vivo models for validation and
reliability of our data. Macaques, the nonhuman primate (NHP) model that closely
resembles the complete spectrum of TB disease and pathogenesis, could serve as
excellent model for evaluating vaccine efficacy before entering the clinical testing
(Dockrell 2016). Thirdly, identification of ideal biomarkers and immune correlates
can be a big boost in assessing vaccine candidates for their potential in inducing -
456 P. Singh et al.
long-term protection and memory response. Thus, an effective vaccine to impede TB

transmission can significantly contribute in TB control and eradication in areas
where TB is highly endemic.
(2) Early Diagnosis
Early and accurate diagnosis of TB is one of the major tools for controlling
transmission of M.tb, especially the drug-resistant strains to uninfected healthy
individuals. A novel and fast diagnostic tool for identification of TB-infected cases
would decrease the rate of transmission in the following three ways:
(a) To have potency in treatment regimen and prevent long-term complications

arising due to no diagnosis or inappropriate diagnosis
Direct smear microscopy and culture of M.tb are standard procedures for TB
diagnosis. While both these techniques are relatively inexpensive, they do have
certain limitations. Direct smear microscopy test is simple and rapid but comes along
with low sensitivity, ranging from 20% to 60% (Steingart et al. 2006). Culturing of
M.tb, which is widely accepted as the gold standard for TB detection, is highly
sensitive but unacceptably slow to provide results, as it takes a minimum of 2 weeks
or as long as 6–8 weeks in case of solid culture techniques (UNITAID 2017). Other
nucleic acid amplification tests (NAAT)-based diagnostic methods have gained
attention in TB diagnosis due to reduced turnaround time with acceptable sensitivity
and specificity. In recent times, commercially available NAATs are being used
globally for TB diagnosis, despite of not being endorsed by the WHO (UNITAID
2017). WHO-endorsed tests include Xpert® MTB/RIF (Xpert), various line probe
assays (LPA), and loop-mediated isothermal amplification (TB-LAMP) (UNITAID
2016 and Nathavitharana et al. 2016). The TB-LAMP test based on isothermal
amplification of M.tb DNA is a low-complexity NAAT which was conditionally
recommended by WHO in 2016 for use in microscopy centers and in high-tiered test
facilities (WHO 2016), but its low-throughput nature limits its application in high-
burden countries where a high-throughput assay is required. It also cannot help in
substituting the rapid NAAT-based tests to detect TB and rifampicin (RIF) resistance
among MDR-TB risk groups. In 2016, an Indian company, Molbio Diagnostics,
introduced Truenat™ for the detection of TB (Truenat™ MTB) as well as for RIF
resistance (Truenat™ MTB-RIF). These are currently under evaluation phase in
India by FIND (UNITAID 2017). A major challenge for delayed TB diagnosis is
nonadherence to treatment regime. Noncompliance to adhere to TB treatment is
mainly attributed to ignorance of the patient in understanding the duration of TB
medicament and significance of completing the entire treatment cycle, nonavailabil-
ity of drugs at the healthcare facility, side effects of TB medications, and the lack of
social support.
(b) Correct and timely diagnosis of TB cases for preventing TB transmission
Delays and inefficiency in diagnosis and treatment of TB can increase the risk of
transmission. GeneXpert is a diagnostic test for TB and RIF resistance that is simple
to perform and provides results within 2 h. However, GeneXpert is not a high-
throughput method, has high maintenance cost, and requires electric supply and
temperature control for operation. These factors pose major challenge to scale up its
use, in resource-limited settings and less developed geographically remote locations
(UNITAID 2016). Light microscopy has a threshold limit of 5000–10,000 bacilli/ml
for detection of M.tb bacilli. However, infectivity dose of M.tb could be as low as
less than 10 bacilli. So, smear-negative patients can also be potent transmitters of the
infection and thus should not be ignored. Although liquid culture is highly sensitive
with a limit of detection as low as 10–100 cfu/ml, it has an extensive detection time
of around 2–6 weeks. Time to detection (TTD) is inversely proportional to bacillary
load and thus a lower TTD means a higher transmission risk (O’Shea et al. 2014). In
the time gap between culturing the bacteria and obtaining the culture positivity
result, the patient is inadvertently transmitting the disease to others. There is an
urgent need to decrease this window period for an overall better control of the
disease.
(c) Prevention in transmission of drug-resistant TB strains
The major reason in emergence of MDR and XDR-TB cases in high-TB-burden

countries is failure of early diagnosis of TB infection. Involvement of private health
organizations such as NGOs to scale up TB control, can raise awareness about drug
resistance and improve access to TB care and treatment. Although assays like
MTBDRplus line probe assay (LPA) provide information on most MDR and
XDR-TB markers, its high sensitivity is impaired due to diligent processing steps
and open hybridization format, thereby leading to amplicon contamination
(UNITAID 2017).
The aforementioned tests suffer from one or the other limitations, including but
not limited to high cost, requirement for maintenance and trained manpower,
dependence on proprietary reagents, supply of constant electricity, and ambient
temperature control, thereby making it unsuitable for resource-limited settings and
remote areas (UNITAID 2017; Trebucq et al. 2011). Therefore, a cost-effective,
rapid, portable, sensitive, and specific diagnostic test with the high-throughput
ability for TB remains an unmet need till this day.
(d) Case Finding/Contact Tracing
The first step in breaking the transmission cycle of TB is early identification of

patients with active TB. Successful TB control depends majorly on case findings and
treatment provision. Routine screening for cases of active TB or latent infection
should be promoted in high-risk populations (active case finding) instead of passive
case finding, i.e., waiting for patients with symptoms to turn up for medical
458 P. Singh et al.
assistance. Investigation on contacts of TB patients helps in limiting transmission.

However, a delay in initiating notable action against TB transmission is always
observed. Earlier studies from countries with high TB incidence have shown that
sociodemographic characteristics, financial status of patients, and fear of stigma of
disease are important determinants that attribute to delay in seeking care from
specialist (American Thoracic Society). Ignorance and superstition about the dis-
ease, its spread and causation, etc. are still prevalent in many areas. The early stage
symptoms in TB are mostly nonspecific. Misinterpretation of active TB symptoms
with other infections such as viral cough and cold has been associated with patient
delay in seeking medical advice. In most of the cases, self-treatment with traditional
home remedies and intake of drugs without supervision of medical practitioner is
usually the first behavioral process in patients. The delay in seeking health supervi-
sion from medical practitioners increases the likelihood of transmission of TB to
contacts. Many TB patients have no knowledge or understanding about
TB. Different notions and myths are associated with TB infection, most of which
are inconsistent with the fact that a bacteria causes TB. Older unwarranted beliefs
which still are prevalent in society include sharing of utensils, food and water
transmitting TB, TB due to hard physical work or exposure to cold or smoke
(Ailinger and Dear 1997; Wandwalo and Mørkve 2000; Lambert and Van der Stuyft
2005; Ayisi et al. 2011), and TB inherited from parents to offspring (Liefooghe et al.
1997; Lin et al. 2007). Misconceptions regarding the knowledge, perception, and
practices of TB exist both at individual and community level (Sharma et al. 2007),
which lead to discrimination and stigmatization of the illness. TB diagnosis is often
associated with social isolation and different psychosocial consequences, which is
dreaded more than the disease itself. These stigmas limit prevention of TB because
at-risk individuals tend to avoid screening and seeking healthcare facilities. This
highlights the need for counseling, creating awareness and motivation, which is
missing in the present-day TB control programs.
(e) Environmental Control/Healthcare Settings
Several environmental factors such as closed indoors, minimal light exposure,

and high humidity favor M.tb to stay viable and infectious in patient’s air droplets,
accelerating transmission to uninfected individuals. Enclosed spaces with poor
ventilation and positive air pressure increase the rate of TB transmission. Other
parameters such as physical proximity and longer and more exposure to infectious
person increases the risk of getting infected. A good quality healthcare service is an
important component at all stages of TB treatment right from diagnosing to drug
resistance screening and to treatment response monitoring. Still, the biggest chal-
lenge is to provide access to quality TB diagnostic services. Available lab services
suffer from different shortcomings like poor infrastructure, limited human resource
capacity, and a weak underlying healthcare system. In many countries, healthcare
facilities are not easily accessible and care is mostly provided by nonspecialized
individuals. Deficiency in specimen collection at district level along with lack of
proper transportation adds on to the challenges. Also, the microscopy centers in
different high-burden countries lack skilled personnel and equipments (Denkinger

et al. 2013). As a consequence, repeated visit at the healthcare facilities seems to be
the common cause of delay in diagnosis and treatment leading toward the nonspe-
cific anti-tubercular.
24.3 Mathematical Modeling
Another approach to break the transmission cycle of TB is mathematical modeling.

This provides an insight to improve the understanding of TB epidemiology and also
helps to strategize TB prevention and control. Various mathematical models were
made by assuming response to TB control, risk of transmission, the incidence of
active cases, and source of infection. A detailed mathematical modeling study of
diagnostic pathway was conducted to estimate the impact of new diagnostic tools
(Trauer et al. 2014). The reduction in occurrence and mortality of TB was observed
by new diagnostic tools against sputum smear microscopy. These models can be
useful for guiding decisions for the development of novel techniques and the effect
of different diagnostic pathways. Another study demonstrated that the change in rate
of contact will help in better estimation of effect of novel diagnostic tests. Expanded
collaboration between policy makers and epidemiologists could prove to be benefi-
cial for future attempts to control TB. Apart from physical and environment
approaches, mathematical modeling has gained considerable interest and serves as
better prediction tool to stop transmission of TB infection, especially in high-burden
countries.
In summary, it is important to determine the major factors responsible for
transmission of TB, as breaking the transmission chain can profoundly decrease
the incidence of TB in high endemic countries. In addition, identifying potential
cases, effective diagnosis, and treatment measures can significantly contribute to
eradicate TB worldwide. The major gaps in our knowledge of understanding the
drivers of transmission of TB need to be addressed effectively in order to curb the
prevalence of TB in high-TB-burden areas.
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Challenges and Advances in TB Drug
Discovery 25
Garima Khare, Prachi Nangpal, and Anil K. Tyagi
Abstract
In this chapter, we provide a comprehensive review of the recent developments
and challenges associated with tuberculosis drug discovery. The chapter begins
with an overview of the global TB burden with an emphasis on the high-burden
countries such as India and the probable reasons associated with high disease
burden. We have discussed the targets for the WHO End TB Strategy along with
the requirements to achieve them. The chapter further provides an insight into the
major obstacles of TB control, the problems associated with the current chemo-
therapy, the need for new anti-TB drugs and expectations from an ideal TB
therapy. The chapter also provides a comprehensive review of the candidate
drugs in the TB drug clinical pipeline with description of their identification,
mechanistic action and in vitro and in vivo efficacy data along with clinical trial
progress. We then provide details about the commonly employed approaches like
whole cell phenotypic approach, target-based virtual screening and repurposing
of drugs for TB drug discovery along with the advantages and major challenges
associated with these approaches. In this regard, the success of whole cell-based
phenotypic screening has been highlighted in view of discovery of the two
recently FDA-approved anti-TB drugs, namely, bedaquiline and delamanid.
The chapter also deals with another promising strategy for TB drug discovery
based on rational drug design with a focus on some of the leads identified by this
Garima Khare and Prachi Nangpal contributed equally with all other contributors.
G. Khare · P. Nangpal
Department of Biochemistry, University of Delhi South Campus, New Delhi, Delhi, India
A. K. Tyagi (*)
Department of Biochemistry, University of Delhi South Campus, New Delhi, Delhi, India
Guru Gobind Singh Indraprastha University, New Delhi, Delhi, India
e-mail: vc@ipu.ac.in

https://doi.org/10.1007/978-981-32-9413-4_25
464 G. Khare et al.
approach. We have also emphasized the recent advancements towards newer

approaches like antisense RNA-based therapeutics, use of natural products, gene-
editing tools such as CRISPR-CAS system and immunotherapy for the develop-
ment of anti-TB molecules. Besides, the chapter also describes the development
of methods to enhance the bioavailability of drugs such as novel delivery systems
like nanoparticles/liposomes and devices for sustained release.
Keywords
Tuberculosis · Mycobacterium tuberculosis · TB drugs · Target-based virtual
screening · Whole cell phenotypic screening · Repurposing approach
Abbreviations
TB Tuberculosis
M. tuberculosis Mycobacterium tuberculosis
RR-TB Rifampicin-resistant tuberculosis
XDR-TB Extremely drug-resistant tuberculosis
MIC Minimum inhibitory concentration
IC Inhibitory concentration
EBA Early bactericidal activity
PK Pharmacokinetic
NTZ Nitazoxanide
LZD Linezolid
NCI National Cancer Institute
AES Allelic exchange substrate
SAR Structure activity relationship
GFP Green fluorescent protein
PS-ODN Phosphorothioate oligodeoxynucleotide
PLG Poly(lactide-co-glycolide)
CRISPR Clustered regularly interspaced short palindromic repeats
Cas CRISPR-associated system
PAM Protospacer adjacent motif
NHEJ Non-homologous end joining
25 Challenges and Advances in TB Drug Discovery 465
25.1 Global TB Scenario
Tuberculosis (TB) is a major global threat to public health. In spite of the presence of
various intervention strategies against TB, the disease continues to persist and leads
to loss of millions of human lives each year. TB is a complex disease due to multiple
outcomes that can be manifested upon infection with the causative agent, Mycobac-
terium tuberculosis. The disease is caused by the inhalation of aerosol droplets
containing the pathogen expelled from a diseased individual by coughing/sneezing.
TB is the ninth leading cause of human deaths worldwide and the leading cause of
deaths from a single infectious agent, ranking above HIV/AIDS (WHO 2017). The
epidemiology of the disease is indeed alarming and requires attention towards its
urgent control. According to the WHO report on tuberculosis, 10.4 million people
developed TB in 2016 worldwide, of which ~1.0 million were HIV positive (WHO
2017). 65% of these total TB incident cases were estimated to be in males and
children accounted for 6.9% TB cases in the year 2016 (WHO 2017). Globally, 1.3
million HIV-negative people died of TB (down from 1.7 million in 2000) with an
additional 0.37 million TB deaths observed in HIV-positive individuals in the year
2016 (WHO 2017). Drug-resistant TB continues to be a major threat with an
estimated 0.6 million new cases resistant to rifampicin (RR-TB) in 2016 at a global
level, of which 0.49 million had multidrug-resistant TB (MDR-TB). There were
about 0.24 million deaths globally from MDR/RR-TB in 2016 (WHO 2017).
It has been observed over the years that India has been one of the highest
TB-burden countries with high rate of incidence, mortality and resistant cases
(WHO 2017). The reason for this can be largely attributed to the nature of the
disease transmission, high population and overcrowding. Moreover, India also has
major problems of malnutrition, poor hygienic conditions, poor supply of drugs and
unregulated use of medicines, which have contributed in a big way to the high
disease burden.
466 G. Khare et al.
WHO End TB Strategy has proposed a target, with reference to the estimates of
2015, to achieve a 95% reduction in TB deaths and a 90% reduction in TB incidence
(new cases per year) by 2035 (Uplekar et al. 2015). However, fulfilment of this goal
demands providing TB care, preventive methods and awareness of health coverage
at global level along with a deliberate collaboration among various stakeholders to
tackle the socio-economic factors related to TB (WHO 2017). Moreover, it is of
utmost importance to develop groundbreaking technological advancements in the
next 5–7 years, whose implementation ought to result in reduction in the TB
incidence rate at a level faster than in the past (WHO 2017).
25.2 Major Obstacles to TB Control
Since the discovery of M. tuberculosis by Robert Koch in 1882, as the causative

agent of TB, the global TB epidemic till date remains persistent, highlighting the
shortcomings of the current control measures available for combating tuberculosis.
The bacteria spreads very easily through aerosols (a few droplet nuclei of 1–5
microns in diameter) which are coughed by an active TB patient and are inhaled
by an uninfected person, and this easy transmission mode poses a big challenge to
curtail the spread of this disease. BCG, the only vaccine available against TB, is
highly effective in preventing childhood tuberculosis, but it is unsatisfactory in
preventing pulmonary tuberculosis in adults showing a variable protective efficacy
ranging from 0% to 80% (Colditz et al. 1994). The current diagnostic tests such as
X-ray, Mantoux test, culture-based test and GeneXpert suffer from several
limitations of being cost ineffective and time consuming and having shortcomings
of sensitivity or specificity. Current efforts are being made towards the development
of a better TB vaccine as well as an easy, rapid and effective diagnostic test.
The current chemotherapeutic regimen for the treatment of drug-susceptible
tuberculosis consists of four first-line drugs, namely, rifampicin, isoniazid,
pyrazinamide and ethambutol, administered for a span of 6 months (Guidelines for
treatment of drug-susceptible tuberculosis and patient care (2017 update). http://
apps.who.int/iris/bitstream/handle/10665/255052/9789241550000-eng.pdf;jsession
id¼86C860DB7117D77D4A072A39ABCD6429?sequence¼1). The standard treat-
ment regimen for drug-susceptible TB comprises of 2 months of initiation phase
consisting of all the four drugs (termed as 2HRZE), followed by a 4-month-long
continuation phase consisting of rifampicin and isoniazid (termed as 4HR) (Guidelines
for treatment of drug-susceptible tuberculosis and patient care (2017 update). http://
apps.who.int/iris/bitstream/handle/10665/255052/9789241550000-eng.pdf;jsessionid
¼86C860DB7117D77D4A072A39ABCD6429?sequence¼1). These drugs are
administered in a daily dosing frequency, and the use of fixed-drug combination
(FDC) has been recommended by WHO over separate drugs (Guidelines for treatment
of drug-susceptible tuberculosis and patient care (2017 update). http://apps.who.int/
iris/bitstream/handle/10665/255052/9789241550000-eng.pdf;jsessionid¼86C860DB
7117D77D4A072A39ABCD6429?sequence¼1). This protracted therapy is one of
the major reasons for the TB patients to default on the therapy, which leads to
non-compliance and non-adherence. Thus, in spite of having an extremely effective

treatment for treating active TB, the non-compliance to the therapy has led to an
inexorable increase in the emergence of drug-resistant strains of the pathogen
leading to drug-resistant TB cases (WHO guidelines for the programmatic manage-
ment of drug-resistant tuberculosis. http://apps.who.int/iris/bitstream/handle/10665/
130918/9789241548809_eng.pdf;jsessionid¼BBA844744A619EF170C00783C6
55E148?sequence¼1; Chiang et al. 2010). This kind of resistance can be classified
as an acquired (secondary) drug resistance; however, primary drug resistance can
also occur by infection with a drug-resistant strain of the pathogen (WHO guidelines
for the programmatic management of drug-resistant tuberculosis. http://apps.who.
int/iris/bitstream/handle/10665/130918/9789241548809_eng.pdf;jsessionid¼BBA
844744A619EF170C00783C655E148?sequence¼1; Chiang et al. 2010). Resis-
tant cases of TB can be divided into various categories depending on the kind of
resistance to isoniazid (isoniazid-resistant TB), to rifampicin (RR-TB), to both
rifampicin and isoniazid (MDR-TB), to any fluoroquinolone and to at least one of
the injectable second-line drugs (amikacin, kanamycin, capreomycin) in addition to
both rifampicin and isoniazid (XDR-TB) (WHO guidelines for the programmatic
management of drug-resistant tuberculosis. http://apps.who.int/iris/bitstream/han
dle/10665/130918/9789241548809_eng.pdf;jsessionid¼BBA844744A619EF170
C00783C655E148?sequence¼1; Chiang et al. 2010). Treatment of the drug-
resistant strains requires the use of various combinations of the second-line drugs
such as levofloxacin, moxifloxacin, gatifloxacin, amikacin, kanamycin,
capreomycin, streptomycin, ethionamide, cycloserine, linezolid and clofazimine.
The therapy for resistant cases may last anywhere from 9 to 24 months. The
emergence of these multidrug-resistant (MDR) strains poses a serious challenge to
the world’s health and towards the global control of this disease. It has been
estimated that an active TB patient undergoing the treatment may transmit the
infection to at least ten uninfected people suggesting the requirement of stringent
control measures (www.who.int/mediacentre/factsheets/fs104/en/). Besides, the
unpleasant side effects and a high pill burden of these drugs make the treatment of
drug-resistant TB and compliance to the therapy an extremely daunting task. The
prevalence of HIV makes the situation even more precarious due to an enhanced
susceptibility of the HIV-infected immunocompromised people to TB infection.
Additionally, the situation is complicated by the difficulty faced by using these
anti-TB drugs along with antiretroviral therapy, which shows negative drug-drug
interactions and, hence, precludes the use of these anti-TB drugs in HIV-positive
patients (López-Cortés et al. 2002). Further, the key challenge is also to treat
individuals who are subclinically infected with M. tuberculosis and are at a lifetime
risk of reactivation TB due to various reasons such as HIV infection, anti-TNF
therapy, diabetes, malnutrition or lowering of immunity (Narayanan et al. 2010;
Gardam et al. 2003; Stevenson et al. 2007). The complexity also arises due to the fact
that the pathogen has the ability to modulate the immune system thereby evading the
immune surveillance. Its ability to persist in a latent and low metabolic state for years
poses significant challenge like drug tolerance. Hence, latent TB disease requires
several months of treatment for complete sterilization.
468 G. Khare et al.
Thus, in view of the above challenges in the TB treatment, it is of utmost

importance to develop novel chemotherapeutic regimens that are able to (i) reduce
the duration of this long drawn therapy, (ii) reduce the pill burden, (iii) target the
latent pathogen, (iv) target drug-resistant strains of the pathogen and (v) can be easily
co-administered with HIV medication.
Although considerable efforts have been made in the field of TB drug discovery
as is evident from a relatively filled drug pipeline now as compared to a few decades
earlier, the progress of TB drug discovery program has been extremely slow with
only two new anti-TB drugs, bedaquiline (marketed as Sirturo) and delamanid,
receiving the FDA approval in the last 50 years that too with limited access and
reserved only for the treatment of MDR-TB. Hence, looking at the high attrition rate
in the TB drug discovery, it is important to develop robust approaches for identifying
better candidate drug molecules that can be channelled for clinical assessment so that
more anti-TB drugs can reach the market.
This chapter provides a comprehensive review of the recent developments in the
field of TB drug discovery and addresses the major challenges associated with the
current approaches being employed for the identification of new drugs.
25.3 TB Drug Clinical Pipeline: The Prospective Future Anti-TB

Drugs
Although the TB drug pipeline remained almost empty for a long period of time till
the 1990s, it is indeed optimistic to see that it is currently filled with more than
20 molecules, which are being assessed under different clinical phases (https://www.
newtbdrugs.org/pipeline/clinical) (Fig. 25.1). Moreover, apart from the molecules in
clinical trials, many more molecules are in the lead optimization phase or in the early
preclinical stages and have the promise of entering the clinical pipeline (Fig. 25.1)
(https://www.newtbdrugs.org/pipeline/clinical).
Fig. 25.1 Candidate anti-TB drugs in various stages of clinical trials
The number of candidate molecules in the TB drug pipeline provides no assur-

ance that these molecules may reach the advance stages of clinical development as is
evident from the fact that only few molecules are presently being evaluated in
phase III and almost 50% are being evaluated as combination regimens of these
drugs. This highlights a high attrition rate and the necessity of more molecules to fill
the pipeline. Besides, the failure of the current candidate molecules reflects the
limitations and caveats of the existing drug discovery approaches and emphasizes
the importance of devising innovative strategies and tools to develop new molecules
or increase the efficacy of molecules that qualify for clinical trials.
The anti-TB molecules currently in the clinical pipeline belong to various classes
like fluoroquinolones, diarylquinolines, nitroimidazoles, benzothiazinones, etc.,
targeting various proteins/enzymes/pathways of M. tuberculosis including cell wall
biosynthesis enzymes, energy metabolism and protein synthesis. Most of these
molecules have been identified either through whole cell phenotypic screening or
by repurposing of the drugs already in use for other diseases. Candidate drugs in TB
pipeline are mentioned below.
25.3.1 BTZ043
The antitubercular agent BTZ043 (belonging to the nitrobenzothiazinone (BTZ)

class) specifically blocks the mycobacterial enzyme decaprenyl-phosphoribose-2-
0
-epimerase (DprE1), responsible for the synthesis of a cell wall component
D-arabinofuranose, and shows MIC in nanomolar range against the members of
the M. tuberculosis complex (Makarov et al. 2009). BTZ043 showed superior
antitubercular activity in comparison to isoniazid in vivo in mouse model with a
470 G. Khare et al.
low toxicological potential and was also well tolerated in rats and mini pigs
(Kloss et al. 2017). The molecule is currently being evaluated in phase I trial
25.3.2 Contezolid (MRX-4/MRX-1)
Linezolid (LZD) belongs to oxazolidinone class of antibiotics that showed potent

in vitro and in vivo activities against M. tuberculosis and was subsequently used in
humans to treat drug-resistant TB; however, its use was restricted due to toxicity
issues, including myelosuppression and peripheral and optic neuropathy (Mehta
et al. 2016; Lee et al. 2012). Contezolid (MRX-1), a new oxazolidinone, was
developed to treat gram-positive infections, such as methicillin-resistant Staphylo-
coccus aureus and vancomycin-resistant enterococci, and showed decreased toxicity
as compared to LZD (Gordeev and Yuan 2014; Shoen et al. 2018). MRX-1 showed
promising in vitro as well as in vivo activity against both drug-susceptible and drug-
resistant M. tuberculosis in mice model and is currently being evaluated in phase III
trials (https://www.newtbdrugs.org/pipeline/clinical, Gordeev and Yuan 2014).
25.3.3 OPC-167832
OPC-167832, a newly synthesized carbostyril derivative discovered by Otsuka

healthcare, inhibits decaprenylphosphoryl-β-D-ribose 20 -oxidase (DprE1), essentially
involved in cell wall biosynthesis of M. tuberculosis (http://www.cptrinitiative.org/
wp-content/uploads/2017/05/Jeffrey_Hafkin_CPTR2017_JH.pdf). This anti-TB com-
pound shows in vitro as well as in vivo efficacy against both laboratory and clinically
isolated strains including multidrug-resistant and extensively drug-resistant
M. tuberculosis (http://www.cptrinitiative.org/wp-content/uploads/2017/05/Jeffrey_
Hafkin_CPTR2017_JH.pdf). Furthermore, OPC-167832, when administered along
with delamanid, showed superior efficacy to the standard regimen RHZE (rifampicin
+ isoniazid + pyrazinamide + ethambutol) in mice (http://www.cptrinitiative.org/wp-
content/uploads/2017/05/Jeffrey_Hafkin_CPTR2017_JH.pdf). OPC-167832 is pres-
ently being evaluated in phase I trial (https://www.newtbdrugs.org/pipeline/clinical).
25.3.4 GSK070
GSK070 is an oxaborole derivative that targets the leucyl-tRNA synthetase

(LeuRS) required for charging the tRNALue with leucine, thereby inhibiting
protein synthesis (Palencia et al. 2016; Rock et al. 2007). GSK70 was observed to
demonstrate in vitro and in vivo efficacy against M. tuberculosis with potent
enzyme inhibition of M. tuberculosis LeuRS (IC50 ¼ 0.216 μM) (Palencia et al.
2016). GSK70 recently qualified for phase I clinical assessment (https://www.
newtbdrugs.org/pipeline/clinical).
25.3.5 Macozinone (PBTZ169)
Lead optimization studies of the compound BTZ043 by using medicinal chemistry

resulted in PBTZ169, which is a piperazinobenzothiazinone derivative. PBTZ169
covalently binds to DprE1, thereby inhibiting cell wall biosynthesis (Trefzer et al.
2010; Makarov et al. 2014). PBTZ169 has an MIC99 against M. tuberculosis in
nanomolar range (0.3 ng/ml) and has shown additive effects with many TB thera-
peutic agents and has also demonstrated synergistic effects with bedaquiline and
clofazimine in preclinical models (Makarov et al. 2014). Innovative Medicines for
Tuberculosis (iM4TB) foundation (Lausanne, Switzerland) has initiated the phase I
clinical study of PBTZ169 to investigate dose-related safety (https://www.
newtbdrugs.org/pipeline/compound/macozinone-mcz-pbtz-169).
25.3.6 TBI-166
Clofazimine, which is a very potent anti-TB compound belonging to

riminophenazine class of drugs, has shown extremely impressive bactericidal and
sterilizing efficacy against TB both in vitro and in mouse models of the disease
(Reddy et al. 1996). However, it has poor solubility and a long half-life which results
in side effects including pronounced skin discoloration (Job et al. 1990; Levy and
Randall 1970). Lead optimization of clofazimine led to the identification of TBI-166,
which has shown improved physicochemical and pharmacokinetic properties with
similar efficacy as the parent compound (Lu et al. 2011). Based on these preliminary,
preclinical and toxicological studies, TBI-166 was approved for phase I clinical trials
25.3.7 TBA-7371
TBA-7371 is a novel molecule belonging to pyrazolopyridone class of inhibitors

(1,4-azaindole series) identified by performing lead optimization studies of an
imidazopyridine compound. This molecule inhibits DprE1
(decaprenylphosphoryl-β-D-ribose 20 -epimerase) by binding to it non-covalently and
shows an IC50 value of 10 nM with an MIC range of 0.78–3.12 μM and demonstrates
efficacy in a rodent model of tuberculosis (Shirude et al. 2013, 2014; Yuan and
Sampson 2018). The TB Alliance has initiated its phase I trial to evaluate its safety,
tolerability, pharmacokinetics and the pharmacokinetic interactions (https://www.
25.3.8 Telacebec (Q203)
Q203 resulted from the lead optimization of imidazo[1,2-a]pyridine amides that

target the respiratory cytochrome bc complex, which in turn disturbs the electron
472 G. Khare et al.
motive force (Pethe et al. 2013; Lu et al. 2018; Kang et al. 2014). The small molecule
Q203 inhibits the growth of MDR and XDR M. tuberculosis clinical isolates in
culture broth medium with MIC in the low nanomolar range as well as shows
therapeutic efficacy in mice (Pethe et al. 2013; Lu et al. 2018; Kang et al. 2014).
In addition, Q203 displayed good pharmacokinetic and safety profiles; hence, it was
qualified for the phase I trial for a dose escalation study which revealed encouraging
results. Phase II trial for early bactericidal activity (EBA) evaluation of Q203 will be
soon initiated in South Africa (https://www.newtbdrugs.org/pipeline/compound/
telacebec-q203).
25.3.9 Sutezolid (Previously Known as PNU-100480)
Sutezolid exhibited increased in vitro as well as in vivo antimycobacterial activity,

when compared with linezolid against both drug-susceptible and drug-resistant TB
and showed improved safety profile highlighting its potential as an anti-TB agent
(Barbachyn et al. 1996; Cynamon et al. 1999; Shaw and Barbachyn 2011; Wallis
et al. 2010, 2011; Alffenaar et al. 2011). In fact, recent studies also showed that the
use of sutezolid along with standard therapy was able to shorten the treatment
duration by preventing relapse, thus, suggesting that sutezolid may have sterilizing
activity against drug-susceptible TB and MDR-TB (Barbachyn et al. 1996; Shaw
and Barbachyn 2011). Sutezolid was well tolerated and safe up to a daily dose of
1200 mg up to 14 days and phase II trials showed early bactericidal activity. Hence,
it is considered that sutezolid may show clinical efficacy in a larger phase II trial
(https://www.newtbdrugs.org/pipeline/clinical, Wallis et al. 2010).
25.3.10 Delpazolid (LCB01-0371)
LCB01-0371 is also a new oxazolidinone compound similar to linezolid with cyclic

amidrazone. In vitro activity of LCB01-0371 was found to be similar to linezolid with
an improved safety profile (Zong et al. 2018; Kim et al. 2017; Jeong et al. 2010).
In vivo activity of LCB01-0371 against systemic infections in mice was also
evaluated, and it was found to be more active than linezolid against these systemic
infections (Zong et al. 2018; Kim et al. 2017; Jeong et al. 2010). It is now in phase II
trial for the evaluation of EBA studies (https://www.newtbdrugs.org/pipeline/clinical).
25.3.11 SQ109
SQ109 is a novel 1,2-ethylenediamine molecule having a novel mechanism of action

targeting MmpL3, which is a mycolic acid transporter required for mycolic acid
incorporation into the M. tuberculosis cell wall (Tahlan et al. 2012; Grzegorzewicz
et al. 2012). SQ109 inhibited the growth of both drug-susceptible and multidrug-
resistant M. tuberculosis strains, including extensively drug-resistant M. tuberculosis
strains (Sacksteder et al. 2012; Protopopova et al. 2005). It also exhibited synergistic
effect with no adverse pharmacokinetic (PK) parameters and also improved the overall
efficacy of the regimen when given along with standard treatment in mice (Sacksteder
et al. 2012; Chen et al. 2006; Nikonenko et al. 2007). Three phase I studies are
completed for SQ109 in the USA along with two phase II studies in Africa in drug-
sensitive TB patients (https://www.newtbdrugs.org/pipeline/compound/sq109).
25.3.12 Auranofin (Brand Name: Ridaura)
Auranofin is a gold complex FDA-approved drug to treat rheumatoid arthritis

(Suarez-Almazor et al. 2000). The drug is able to reduce and improve arthritis-
related symptoms like pain, tender and swollen joints and morning stiffness.
Auranofin was identified by employing a cell-based screen under nutrient-
deprivation conditions against M. tuberculosis (Harbut et al. 2015). It exhibits potent
inhibition of the growth of both replicating and nonreplicating M. tuberculosis,
which was found to be bactericidal in nature (Harbut et al. 2015). Phase II trial is
initiated to study the efficacy of the auranofin against M. tuberculosis (https://www.
25.3.13 Levofloxacin
Levofloxacin is a second-generation fluoroquinolone, which shows enhanced activ-

ity against gram-positive pathogens, including S. pneumoniae and S. aureus, and
was shown to be effective in the treatment of upper and lower respiratory tract
infections in adults (Peterson et al. 2009; Alsultan et al. 2015). Levofloxacin is one
of the essential medicines listed by World Health Organization and may be used for
the treatment of tuberculosis (https://www.newtbdrugs.org/pipeline/compound/
levofloxacin). TBTC 32/NIAID OPTI-Q phase II studies will determine the
levofloxacin dose and exposure required to achieve the maximal reduction in
M. tuberculosis burden in Peru and South Africa (https://www.newtbdrugs.org/
pipeline/compound/levofloxacin).
25.3.14 Nitazoxanide (NTZ)
NTZ is a synthetic nitrothiazolyl salicylamide prodrug that is deacetylated in the

gastrointestinal tract to the active metabolite tizoxanide and is approved for the
treatment of giardiasis and cryptosporidiosis (Aslam and Musher 2007). NTZ showed
activity against other protozoa, helminths, rotavirus and hepatitis C (Aslam and
Musher 2007; Stachulski et al. 2011; Adagu et al. 2002; Theodos et al. 1998; Darling
and Fried 2009; Korba et al. 2008; Rossignol et al. 2008, 2010). It was also shown to
kill both replicating and nonreplicating M. tuberculosis with a MIC of 16 μg/ml
(de Carvalho et al. 2009). No resistant mutants were found on treatment of
474 G. Khare et al.
M. tuberculosis with various concentrations of NTZ suggesting that NTZ may have
multiple targets (de Carvalho et al. 2009). It is currently undergoing phase II efficacy
trial (https://www.newtbdrugs.org/pipeline/clinical).
25.3.15 Combination Regimens
The drugs that have successfully completed the phase II efficacy trials are now being
evaluated in combination regimens in phase III trials to evaluate their therapeutic
efficacy in shortening the treatment regimen.
25.3.16 Bedaquiline
Bedaquiline belongs to the class of diarylquinolines with a novel mechanism of

action targeting the mycobacterial ATP synthase (Andries et al. 2005; Koul et al.
2007). It was found to exhibit activity against both drug-susceptible and drug-
resistant strains of the pathogen having a strong bactericidal and sterilizing
properties (Andries et al. 2005; Diacon et al. 2009). Bedaquiline got US FDA
approval in 2012 as an anti-TB drug; however, its usage is reserved for the treatment
of MDR-TB cases only (https://www.newtbdrugs.org/pipeline/compound/
bedaquiline-0). It is now being evaluated in various combination regimens in
which bedaquiline and pretomanid will be administered along with existing and
repurposed anti-TB drugs for the treatment of biologically confirmed pulmonary
multidrug-resistant TB (MDR-TB) (https://www.newtbdrugs.org/pipeline/com
pound/bedaquiline-0). Besides, another trial is scheduled to be carried out to assess
its efficacy, when administered with delamanid (https://www.newtbdrugs.org/pipe
line/compound/bedaquiline-0).
25.3.17 Rifapentine
Rifapentine is a semisynthetic derivative of rifamycin family with MIC99 value of

0.25 μg/ml in liquid medium (Sensi et al. 1959; Bemer-Melchior et al. 2000). It is
currently approved for intermittent dosing in the treatment of TB. Also, it is currently
included in combination regimen for phase III trials to test whether these regimens
can shorten the treatment duration (https://www.newtbdrugs.org/pipeline/com
pound/rifapentine). The regimens being evaluated are (i) a single replacement of
rifampin with rifapentine, initial 2 months of isoniazid, rifapentine, ethambutol and
pyrazinamide, followed by another 2 months of isoniazid and rifapentine, and (ii) a
double replacement of rifampin with rifapentine and ethambutol with moxifloxacin –
initial 2 months of isoniazid, rifapentine, moxifloxacin and pyrazinamide, followed
by another 2 months of isoniazid, rifapentine and moxifloxacin (https://www.
newtbdrugs.org/pipeline/compound/rifapentine).
25.3.18 Delamanid
Delamanid (OPC-67683, Deltyba®) belongs to bicyclic nitroimidazole class of

compounds, which showed a marked antituberculosis activity in vitro and a superior
therapeutic efficacy in the chronic mouse model (Matsumoto et al. 2006; Tsubouchi
et al. 2016). In 2014, European Medicines Agency (EMA) approved delamanid for
the treatment of adult pulmonary MDR-TB (Yuan and Sampson 2018). Delamanid-
resistant mutants revealed that it inhibits genes involved in F420-dependent
deazaflavin nitroreductase bioactivation pathway (Fujiwara et al. 2018). In addition,
in a phase IIb global trial, delamanid was found to increase the rate of 2-month
sputum culture conversion, when it was added to an already optimized background
regimen for the treatment of MDR-TB patients (Diacon et al. 2011; Gler et al. 2012).
Clinical studies also revealed the efficacy of delamanid containing regimens in
highly resistant TB patients that included cases with extensively drug-resistant TB
(Skripconoka et al. 2013). Currently, for the evaluation of safety and efficacy of
delamanid at a 200 mg oral daily dose, a phase III trial is being conducted (https://
www.newtbdrugs.org/pipeline/compound/delamanid-0). Moreover, combined
usage of delamanid and bedaquiline is also in progress to evaluate whether their
combination can enhance the efficacy against MDR-TB (https://www.newtbdrugs.
org/pipeline/compound/delamanid-0).
25.3.19 Clofazimine
Clofazimine, as described above, shows potent antitubercular activity (Reddy et al.

1996; Xu et al. 2012) and is now being evaluated in phase III trials for its efficacy,
safety and tolerability, when it is administered in various regimens such as (i) TMC207
plus PA-824 plus pyrazinamide plus clofazimine, (ii) TMC207 plus PA-824 plus
clofazimine, (iii) TMC207 plus pyrazinamide plus clofazimine and (iv) clofazimine
alone, in adult patients with newly diagnosed, smear-positive pulmonary tuberculosis
(https://www.newtbdrugs.org/pipeline/compound/clofazimine).
25.3.20 Pretomanid-Moxifloxacin-Pyrazinamide Regimen
Pretomanid (PA-824, Pa) belongs to nitroimidazo-oxazine class of compounds,

which shows a very potent MIC of 0.125 μg/ml against M. tuberculosis and shows
bactericidal activity during the initial and continuation phases of treatment in murine
model with no issues of cross-resistance with other existing TB drugs (Stover et al.
2000; Tyagi et al. 2005). In addition, combination of PA-824, moxifloxacin and
pyrazinamide (PaMZ) cured mice more rapidly than the first-line regimen of rifam-
pin, isoniazid and pyrazinamide (Nuermberger et al. 2008; Tasneen et al. 2011).
PaMZ represents the first regimen to undergo clinical evaluation for multidrug TB
treatment and has shown very positive results in phase II trial to conclude that it has
476 G. Khare et al.
potential of curing both the susceptible and resistant TB (https://www.newtbdrugs.org/

pipeline/regimen/pretomanid-moxifloxacin-pyrazinamide-regimen). Besides, PaMZ
also had better co-administration ability with the antiretrovirals, which is useful as an
improved treatment option for HIV-TB co-infected patients (https://www.newtbdrugs.
org/pipeline/regimen/pretomanid-moxifloxacin-pyrazinamide-regimen). TB Alliance is
now focusing on advancing the BPaMZ regimen consisting of bedaquiline, PA-824,
moxifloxacin and pyrazinamide (https://www.newtbdrugs.org/pipeline/regimen/
pretomanid-moxifloxacin-pyrazinamide-regimen).
25.4 Advantages and Pitfalls of Various TB Drug Discovery

Approaches
25.4.1 Target-Based Virtual Screening
The growing burden of antibiotic resistance propelled research towards the develop-
ment of new antibiotics resulting in various drug discovery approaches. The genome
sequence of M. tuberculosis was elucidated in the year 1998 (Cole et al. 1998),
which gave a tremendous boost to the approaches such as target-based virtual
screening, generation of gene knockout strains for the identification and validation
of drug targets and system biology, etc. Target-/structure-based virtual screening
relies on the use of computer-based software to identify potential inhibitors against
the target structure by docking a library of small molecules into the active site and
shortlisting the best binding molecules. An important prerequisite for this approach
is the availability of a three-dimensional structure of the target protein, which gained
its pace after the determination of genome sequence of M. tuberculosis. Another
crucial step involved in virtual screening is the identification of an important drug
target, which plays a key role in the pathogenesis of M. tuberculosis. This became
possible as the knowledge about M. tuberculosis genes increased with the availabil-
ity of its genome sequence, which also aided in developing gene deletion mutants.
These knockout strains are then evaluated for their ability to grow in vitro, inside
macrophages and subsequently in animal models for final validation; however, the
major limitation of this technique, for a long time, has been a low frequency of site-
specific recombination making it difficult to obtain the mutants and the inherent
challenge of slow growth rate of M. tuberculosis. With newer methods of
recombineering that were subsequently developed based on linear allelic exchange
substrate, transposon mutagenesis and mycobacteriophage systems, generating
knockout strains in M. tuberculosis became easier and the validation of essential
and important drug targets gained a much faster pace (Van Kessel and Hatfull 2007;
Sassetti et al. 2001). A study by Sassetti et al. revolutionized the drug discovery
efforts by identifying several M. tuberculosis genes required for the in vitro and
in vivo growth of the pathogen by using transposon mutagenesis, which led to a huge
list of important drug targets along with the information on their essentiality for the
growth of the pathogen (Sassetti et al. 2001; Sassetti and Rubin 2003). For instance,
enzymes belonging to energy metabolism were shown to be essential for the growth
of M. tuberculosis by Sassetti et al., and this information led to the identification of
Q203 as an important small molecule inhibiting the cytochrome bc1 complex, which
is currently being evaluated in clinical trials (https://www.newtbdrugs.org/pipeline/
clinical, Kang et al. 2014). Additionally, many drug targets including
UDP-galactopyranose mutase, 2C-methyl-D-erythritol 4-phosphate pathway and
enzymes belonging to purine nucleotide biosynthetic pathway (GuaB2) were also
shown to be essential by transposon mutagenesis by Sassetti et al. and have been
employed as drug targets (Kincaid et al. 2015; Eoh et al. 2009; Singh et al. 2017). In
addition, many other deletion mutants have been developed by using the above-
mentioned methods, which have helped in the identification of several drug targets
including MbtE, SapM, BioA, PptT, DrpE1, Rv3484, etc. (Reddy et al. 2013; Puri
et al. 2013; Kar et al. 2017; Leblanc et al. 2012; Crellin et al. 2011; Malm et al.
2018). For instance, the mbtE mutant was developed by using linear AES method of
homologous recombination, and MbtE was shown to be essential for the virulence
and survival of the pathogen in broth culture and in macrophages. Besides, the
mutant was also shown to be attenuated for its growth in guinea pigs as infection
with it exhibited significantly reduced bacillary load and histopathological damage
in the organs, in comparison to M. tuberculosis-infected animals (Reddy et al. 2013).
Target-based virtual screening has been employed extensively since the last
decade, and till date many compounds have been screened against many important
drug targets. A screening effort that evaluated 20,000 compounds against FtsZ,
which plays an essential role in cell division, led to the identification of the inhibitor
297F, which showed inhibitory activity against M. tuberculosis in vitro growth (Lin
et al. 2014). In another study, inhibitors were identified against M. tuberculosis
thiamine phosphate synthase, an enzyme involved in the biosynthesis of thiamine by
employing virtual screening resulting in promising inhibitors with IC50 of 34 μg/ml
and MIC99-6 μg/ml (Khare et al. 2011). In a recent study by Singh et al., structure-
based virtual screening was employed against the active site of BioA, an enzyme
involved in biotin biosynthetic pathway, which is essential for M. tuberculosis
in vivo survival. It resulted in a few hits with the most potent hit displaying
an MIC90 of 20 μg/ml (Singh et al. 2018). In another study, virtual screening
was carried out by employing NCI library at the active site of M. tuberculosis
40 -phosphopantetheinyl transferase (PptT), which is involved in
phosphopantetheinylation of several important proteins in a post-translational man-
ner (Rohilla et al. 2018). This study led to the identification of a number of molecules
with potent inhibition of the PptT enzymatic activity (IC50 10 μg/ml). Further, by
employing a structure similarity approach based on chemoinformatics, a potent
analogous molecule was identified with IC50 of 0.25 μg/ml, MIC90 of 10 μg/ml
and negligible cytotoxicity (Rohilla et al. 2018). Recently, a study by Rohilla et al.
showed that virtual screening of NCI library against IdeR, an essential iron
478 G. Khare et al.
regulatory transcriptional factor of M. tuberculosis, identified potent hits exhibiting

IC50 values of 1–2 μg/ml (Rohilla et al. 2017). Major advantage of target-based
screening is that there is a prior knowledge about the structure of the compound in
question and the drug target, which makes it easier to elucidate the mechanism of
action of the inhibition observed. Besides, the structural as well as the chemical
knowledge about the compound helps in performing better lead optimization studies
to obtain an improved antitubercular compound.
One of the major limitations of this strategy is the determination of the crystal
structure of many M. tuberculosis proteins as they usually do not express well in
E. coli and remain insoluble when expressed. Methods such as comparative homol-
ogy modelling provide an alternative for the absence of crystal structures; however,
they may not always result in potent inhibitors. Additionally, although this strategy
holds promise in identifying a potent inhibitor against the target, many resulting
enzyme inhibitors are unable to show a good MIC value in vitro (Singh et al. 2018;
Rohilla et al. 2017, 2018; Kumar et al. 2017). M. tuberculosis is a complex pathogen
comprising of a very strong cell wall which hampers the entry of most of the small
inhibitor molecules, which are screened and shortlisted by employing target-based
virtual screening. Even though the shortlisted molecules that are identified by
screening chemical libraries against a validated target show high binding affinity
for the target, their inability to enter the cell due to highly impermeable and
hydrophobic cell wall of the pathogen might prevent them from arresting the cell
growth. Besides, efflux of the compounds by the mycobacterial cells may also
contribute to their poor MIC values (Kumar et al. 2017; Rodrigues et al. 2012;
Zuniga et al. 2015; Kanji et al. 2016; Zhang et al. 2017; Parthasarathy et al. 2016).
For instance, target-based virtual screening against many targets including PimA and
PanC, both shown to be essential drug targets, resulted in the identification of several
hits by screening many chemical libraries; however, none of the shortlisted
molecules showed cellular activity emphasizing the possibility of their lack of
entry into the cell (Kumar et al. 2017). Moreover, since M. tuberculosis is highly
lipid rich, it is expected that a more lipophilic molecule will have a better permeabil-
ity inside the mycobacterial cells and thus may be more potent as it has also been
seen in the case of current anti-TB drugs, which are peculiarly more lipophilic than
inhibitors against other bacteria (Kumar et al. 2017; Piccaro et al. 2015; Machado
et al. 2018). However, in medicinal chemistry, the physicochemical characteristics
such as high lipophilicity are not considered favourable for the development of a
molecule into drug. This may explain the high attrition rate in the virtual screening
approach with no identified compound reaching the TB drug pipeline, since most of
the libraries selected for the screening are chosen as per the existing drug-likeness
rules and exclude the lipophilic molecules. Hence, it seems more logical to recon-
sider the drug-likeness rules in the case of TB drug discovery, and new thinking
beyond the existing dogma of Lipinski’s rule of five is needed to succeed (Piccaro
et al. 2015; Machado et al. 2018).
25.4.2 Whole Cell Phenotype Screening
The fact that none of the molecules that are being evaluated in clinical pipeline are
derived from structure-based screening, there was a paradigm shift from computa-
tional methods to whole cell phenotypic screening approach for the identification of
new anti-TB agents having novel mechanism of action. The method involves direct
screening of small molecule libraries against the growth of M. tuberculosis in broth
culture. The use of this approach gained pace after the discovery of bedaquiline
(TMC207) and the successful identification of its target ATP synthase through
isolation of resistant colonies and whole genome sequencing (Andries et al. 2005).
Moreover, the success of this strategy is evident from the number of drugs in the
clinical pipeline, which have been identified by whole cell phenotypic screening.
Apart from bedaquiline (TMC207), various molecules such as SQ109, OPC-67683,
PBTZ-169 and Q203 have been identified by employing this approach exhibiting
potent inhibition of M. tuberculosis growth and are in various clinical, preclinical or
early drug discovery stages (Makarov et al. 2014; Pethe et al. 2013; Lu et al. 2018;
Sacksteder et al. 2012; Protopopova et al. 2005; Matsumoto et al. 2006; Tsubouchi
et al. 2016). The power of this approach is that it circumvents the problems
associated with target-based screens like compound penetration, target redundancy,
etc.; however, a major drawback of this approach is the lack of knowledge about the
mode of action of the drug, and hence, no inputs can be obtained via structure
activity relationship studies and medicinal chemistry for better and improved drug
designing (Zuniga et al. 2015). In fact, many times, for example, in the case of
multiple targets, it is not possible to isolate resistant colonies which makes target
identification a hard task. However, genomic tools for the identification of mutations
in resistant colonies and transcriptional profiling studies in the presence and absence
of drug along with the advent of proteomics and metabolomics have been shown to
be promising and valuable tools for finding the drug targets. In addition, if one is able
to identify a potent inhibitor by using whole cell approach, it is sometimes difficult to
understand its inhibitory activity in vivo due to a number of microenvironments the
bacteria faces including hypoxic conditions, oxidative stress, acidic stress as well as
the various metabolic states that the pathogen can acquire (Kumar et al. 2017; Koul
et al. 2011). Hence, the development of better screening methods like carbon
starvation model, hypoxic model and replicating and nonreplicating
M. tuberculosis model systems that can mimic and represent the in vivo situation
that M. tuberculosis encounters in human host will provide hope for improved drug
designing (Kumar et al. 2017; Sala et al. 2010; Koul et al. 2008). More recently, TB
investigators are focusing towards developing rapid and faster methods for screening
inhibitors directly against the intraphagosomal growth of M. tuberculosis inside
macrophages, the host niche where the bacteria resides, by employing GFP
expressing strains of M. tuberculosis (Khare et al. 2013).
480 G. Khare et al.
25.4.3 Screening of Natural Products
The limited chemical space and diversity provided by screening libraries of small
molecules result in the identification of hits with limited target diversity, which also
highlights why certain pathways are always the frequent hits (Kumar et al. 2017).
Hence, considerable attention has been drawn towards the use of natural products as
starting point for TB drug discovery that can provide diverse chemical space with
novel scaffolds, which are not present in the pharmaceutical libraries used for target-
based as well as whole cell screening methods (Kumar et al. 2017; Cragg and
Newman 2013). Natural products from medicinal plants or antibacterial bioactive
compounds have shown promising results in terms of growth inhibitory potential
against M. tuberculosis (Rodrigues Felix et al. 2017; Hartkoorn et al. 2012;
Pruksakorn et al. 2010; Steinmetz et al. 2007). These include secondary metabolites
isolated from plants and other microbial sources such as bacteria, fungi, marine
organisms and algae, belonging to various chemical types such as terpenes
(sesquiterpenes, diterpenes, sesterterpenes, triterpenes), steroids (sterols), alkaloids
(indole, quinoline, pyridoacridone, manzamine alkaloids, etc.) and aromatics
(flavonoids, chalcones, coumarins, lignans, xanthones, anthracenes, anthraquinones,
naphthalenes, chromones, etc.). Apart from the secondary metabolites, polyketides
(acetylenic fatty acids, polycyclic esters, quinones, etc.) and peptides have also
shown growth inhibitory potential. Moreover, these metabolites are bioactive in
nature; their relative bioavailability is quite high which thereby increases their access
to the site of action (Quan et al. 2017). One of the naturally occurring classes of
compounds is phenazines that are biosynthetically produced by Actinobacteria
phylum, which are used against bacteria and fungi as broad-spectrum antibiotics
(Quan et al. 2017; Laursen and Nielsen 2004). There is a renewed interest in
considering riminophenazines as lead compounds for TB drug discovery, especially
after clofazimine belonging to the same class was shown to work very effectively
against MDR-TB, when administered in combination with gatifloxacin, ethambutol,
pyrazinamide, prothionamide, kanamycin and high-dose isoniazid for 9 months
(Reddy et al. 1999; Van Deun et al. 2010). However, because of the toxicity
observed with the use of clofazimine, analogues of this natural product were
developed, and as mentioned above, TBI-166 has been identified as a promising
compound (https://www.newtbdrugs.org/pipeline/clinical, Reddy et al. 1996; Job
et al. 1990; Levy and Randall 1970; Lu et al. 2011). Likewise, piperidines is another
class of naturally available molecules isolated from black pepper, which have been
used as wide range of drugs such as neuroleptics, vasodilators, antipsychotics and
opioids (Quan et al. 2017). SQ109, the drug that is currently in the clinical pipeline,
is an adamantine-containing hydroxydipiperidine that exhibits potent growth inhibi-
tory property against M. tuberculosis in vitro and in vivo (Sacksteder et al. 2012;
Protopopova et al. 2005). Similarly, BTZ043 also belongs to piperidine-containing
benzothiazinone displaying inhibitory activity against the clinical isolates of
M. tuberculosis and drug-resistant strains (Makarov et al. 2009; Kloss et al. 2017;
Quan et al. 2017). Notably, both SQ109 and BTZ043 were identified through
screening of dipiperidines and sulphur containing heterocycle libraries, respectively,
emphasizing the strength of using natural products for the identification of
antitubercular compounds given that these metabolites/peptides comprise of broad
plethora of diverse scaffolds and pharmacophores. Moreover, other molecules in the
clinical pipeline also belong to various classes of secondary metabolites like mycins
and quinolones. However, the reasons for apprehension in employing this approach
and a lag observed in the success of using these natural products are (i) difficulties in
extraction of high yields of purified compounds, (ii) complexity in determining the
structure of the compound, (iii) accessibility of the source material reproducibly,
(iv) lack of safety studies and (v) lack of information about mechanism of action.
Hence, innovative research is required to overcome these shortcomings.
25.4.4 Repurposing of Drugs
Another important strategy that has a lot of translational scope, which has gained
focus and has resulted in promising molecules currently in the clinical pipeline, is the
repurposing of the existing drugs also known as therapeutic switching or
repositioning approach (Fig. 25.1) (Maitra et al. 2015). Repurposing drug approach
employs the use of already existing drugs against various other diseases. Some of the
drugs that are used to treat a particular condition can also interact with some other
important target(s) and show its effect, which provides a window to analyse its
therapeutic efficacy. Repurposing of the known drugs is promising as it is less time
consuming in terms of the translation from preclinical work to the market, is less
risky and is also less costly (Fig. 25.2).
This approach benefits from the fact that these molecules are already well
characterized in the context of its target validation, hit-to-lead optimization, in vivo
metabolic studies and their safety and toxicity profiling (Maitra et al. 2015). Only 1 in
482 G. Khare et al.
Fig. 25.2 The figure depicts various steps involved in the most commonly used drug discovery
approaches for the identification of new anti-TB drugs
10,000 new chemical entities entering into pharmaceutical research actually makes it
to the market; hence, the compounds already found to be safe in early-stage trials are
less risky to begin with. It takes minimum a decade for a non-repurposed drug to reach
the market as compared to only ~4 years for a repurposed drug. The most significant
example of this approach is the use of sildenafil, which was used as an antihyperten-
sive drug, also been shown to shorten the TB treatment in mouse model studies, when
used along with standard drug regimen (Maiga et al. 2012). Moreover, three drugs,
namely, clofazimine, linezolid and moxifloxacin, currently present in the TB drug
clinical pipeline have resulted from repurposing approach (Van Deun et al. 2010; Till
et al. 2002; Yanagihara et al. 2002; Alvirez-Freites et al. 2002). Clofazimine was
initially used to treat leprosy and was shown to be successful in treating MDR- and
XDR-TB; however, due to its side effects, it is being evaluated in combination with
other TB drugs and further analogues are being prepared and tested such as TBI-166
(https://www.newtbdrugs.org/pipeline/clinical, Reddy et al. 1996; Job et al. 1990;
Levy and Randall 1970; Lu et al. 2011; Xu et al. 2012; Van Deun et al. 2010; Garrelts
1991). Fluoroquinolones are potent broad-spectrum antibiotics that inhibit
topoisomerases II and IV, in turn inhibiting DNA replication (Drlica and Zhao
1997). Moxifloxacin and gatifloxacin, which are the new generation fluoroquinolones,
have shown sterilizing properties against M. tuberculosis in both in vitro and in vivo
studies and are currently being used as second-line treatment for TB (Alvirez-Freites
et al. 2002). Moxifloxacin is now being tested in phase III trials in combination with
other drugs to evaluate its efficacy in shortening the treatment regimen (https://www.
newtbdrugs.org/pipeline/clinical). Other classes of drugs such as members of the
avermectin family, which are used for treating helminthic infections, are being tested
for their activity against M. tuberculosis (Lim et al. 2013).
Efforts have also been made by the Indian scientists towards development of new
drugs by using the repurposing approach. For example, Singhal et al. showed that the
FDA-approved drug metformin used for treating diabetes was able to inhibit the
intracellular growth of M. tuberculosis and enhance efficacy of the existing first-line
TB drugs, thereby suggesting its use as part of adjunctive TB therapy (Singhal et al.
2014). Brindha et al. carried out virtual screening of FDA-approved drugs against
the potential targets of M. tuberculosis, namely, TrpD and CoaA, and further
screened the top ranking molecules for their ability to inhibit the in vitro growth of
M. tuberculosis resulting into the identification of two potential inhibitors of suscep-

tible as well as resistant strains of M. tuberculosis, namely, lymecycline and
cefpodoxime (Brindha et al. 2017). Moreover, lymecycline and cefpodoxime
exhibited synergistic activity with rifampin and isoniazid against M. tuberculosis,
which suggests the potential of these drugs for the treatment of tuberculosis (Brindha
et al. 2017). In another study, it was shown that administration of verapamil, an
efflux pump inhibitor, as part of adjunctive therapy to infected mice was able to
shorten the duration of standard TB regimen by accelerating the bacterial clearance
and lowering down the relapse rates in comparison to mice that received only
standard chemotherapy (Gupta et al. 2013). In addition, when verapamil was
administered along with bedaquiline and clofazimine, it was able to sharply decrease
the MIC of these drugs by 8- to 16-fold (Gupta et al. 2014). Another drug, namely,
statin, which is used for lowering down of blood cholesterol, when given along with
the first-line antitubercular drugs reduces the lung bacillary load in chronically
infected mice (Dutta et al. 2016). Thus, drug repurposing approach is a very
attractive strategy and provides alternatives for the treatment of drug-resistant cases.
25.4.5 New Approaches
a. RNA-Based Therapeutics
Apart from the major challenges associated with the TB chemotherapy, the biggest
concern that arises in developing new anti-TB agent is the emergence of drug
resistance. Even if a new molecule is identified as a potent anti-TB agent targeting
484 G. Khare et al.
a novel protein or metabolic pathway, the problem of developing drug resistance

against the newly identified drug still prevails, and thus, cutting-edge research and
innovative technologies are required, which can circumvent the problem of emer-
gence of resistant strains. One such strategy is the use of RNA-based therapeutics
such as antisense oligonucleotides, which may represent antitubercular compounds
of the future. Antisense oligonucleotides complementary to the target mRNA
sequence are designed which lead to the formation of target mRNA/antisense
oligo duplex (Bai and Luo 2012). This duplex recruits RNaseH and cleaves the
target mRNA molecule resulting in silencing of the target gene (Bai and Luo 2012).
One of the limitations, however, associated with the use of oligonucleotides can
be their unstable nature and inefficient delivery. Recent studies have shown
that modified oligonucleotides such as phosphorothioate oligodeoxynucleotide
(PS-ODNs) have enhanced cellular stability, and the use of nanoparticles or
liposomes results in better delivery, stability and increased bioavailability. In a
study by Meng et al., a new formulation, which involved the use of anionic liposome
for encapsulation and delivery of mecA-specific PS-ODNs to target methicillin-
resistant Staphylococcus aureus, was employed (Meng et al. 2009). Infected mice
when treated with the encapsulated PS-ODN833 downregulated mecA and rescued
the animals from MRSA-caused septic death (Meng et al. 2009). Harth et al.
investigated the effect of hairpin loop extensions by using a sequence-specific
PS-ODNs having 30 and 50 hairpin loop extensions targeting the 30–32 kDa protein
complex (antigen 85 complex) that is involved in the mycolic acid synthesis of
M. tuberculosis (Harth et al. 2007). These PS-ODNs with hairpin loop extensions
inhibited bacterial growth in broth culture, inside human macrophages, and also
reduced target gene transcription by >90% and showed increased bacterial sensitiv-
ity to isoniazid (Harth et al. 2007). Although very preliminary studies have been
conducted by employing antisense RNA as a therapeutic tool against
M. tuberculosis, due to promising results witnessed in these studies, further research
is needed to take this approach forward.
b. Delivery Systems
b1. Nanoparticles/Liposomes
Recent studies are also being focused on developing better delivery strategies to
increase the bioavailability of the anti-TB agents. A drug either administered via
intravenous route or given orally gets distributed throughout the body, and hence,
there are a limited number of molecules that reach the target site (Nasiruddin et al.
2017). Moreover, there can be non-specific or adverse side effects as well. In the case
of mycobacterial infection, the drugs need to further reach the bacteria residing in
macrophages and inside granulomas. Besides, the short half-life and rapid clearance
of the drug also limit its effectiveness (Greenblatt 1985). Hence, to overcome this
challenge, either we need very effective drugs or better delivery methods that can
enhance the effectiveness of the drug. To this end, nanoparticles or liposomal

preparations encapsulating the TB drugs are being developed. Nanoparticles are
taken up very efficiently by cells and have the property of controlled, slow and
persistent drug release making them an attractive and promising tool for drug
delivery. Many modified nanoparticles have been prepared, for example, PEGylated
nanoparticles to increase the bioavailability of drugs (Pandey et al. 2003; Sharma
et al. 2004). In a study, a single subcutaneous injection of PLG nanoparticles
encapsulated with the first-line TB drugs, rifampicin, isoniazid and pyrazinamide
resulted in sustained plasma drug levels for 32 days and in lungs and spleen for
36 days (Pandey and Khuller 2004). This led to complete sterilization of the organs
of M. tuberculosis-infected mice and demonstrated better therapeutic efficacy as
compared with daily oral intake of free drugs (Pandey and Khuller 2004). Liposomes
offer an inherent advantage that they have fusogenic abilities to fuse with the
macrophages and can efficiently release the drugs into the macrophage cells,
which is the primary niche of mycobacteria. In fact, target-based delivery systems
are also being developed to avoid non-specific interactions of the drug-encapsulated
nanoparticles or liposomes by decorating them with molecules such as mannose
residues or O-SAP (O-stearyl amylopectin), which can specifically target the
macrophages (Mahajan et al. 2010; Vyas et al. 2004). Thus, the superior drug
bioavailability can help enhance its therapeutic usefulness even at low doses of the
formulation, which may further help in reducing the period of chemotherapy and
patient’s compliance. In a study by Deol et al., it was demonstrated that liposome-
encapsulated anti-TB drugs, isoniazid and rifampicin, showed better efficacy than
free drugs against tuberculosis in mice model (Deol et al. 1997). Thus, advances
should also be made towards improving various delivery systems, which can help in
slow and sustained release of the drugs to finally increase the effectiveness and the
associated therapeutic efficacy.
b2. Devices for Sustained Release
One of the problems associated with the control of TB relates to poor adherence
of the patients to the lengthy chemotherapy, which requires daily administration,
high pill burden and frequent dosing. Hence, current research is also being focused
on the development of orally administered devices, which can help in increasing the
bioavailability of the drug along with sustained release, with holding capacities for
close to a month’s pill dosage (Caffarel-Salvador et al. 2017). Such devices would
make it feasible to provide the patient with the one time-large dose along with
controlled release systems, which would help in getting rid of the needle injections
and its associated complications. A similar device having these properties and ability
to go and reside inside the GI tract is currently under preclinical evaluation. Such
devices are advantageous over the existing injectable or oral drugs because of the
ease of administration, a low immunological response and a greater accommodation
of the drug inside the GI tract (Caffarel-Salvador et al. 2017). These devices would
486 G. Khare et al.
lead to drastic increase in the patient’s adherence to the therapy, which would prove
a boon for the prevention of drug-resistant TB cases.
c. Gene-Editing Tools
Recombineering techniques based on homologous recombination for the generation

of deletion mutants are not a very efficient way to validate important drug targets due to
high frequency of illegitimate recombinations and time-consuming procedure. Besides,
multiple steps and specialized reagents are required, which make the procedure cost
ineffective. As identification and validation of essential genes is a crucial requirement for
the discovery of novel molecules, newer tools are needed that can speed up this process.
CRISPR/CAS technology provides an alternative to the conventional method of gene
silencing and has proven to be very useful in M. tuberculosis (Choudhary et al. 2015;
Singh et al. 2016). The clustered regularly interspaced short palindromic repeats
(CRISPR)/CRISPR-associated (Cas) system is a novel genome-editing tool found in
bacteria and archaea responsible for the adaptive immune system of prokaryotes
providing them with resistance to invading foreign viruses or plasmids (Makarova
et al. 2011). The type II CRISPR/Cas system comprises of two short RNA and the
DNA endonuclease Cas9. The short RNAs direct Cas9 DNA endonuclease to the target
DNA sequence called the protospacer on the target DNA next to the protospacer
adjacent motif (PAM) for site-specific cleavage resulting in double-stranded breaks,
which can be repaired either by (i) the efficient, however, error-prone non-homologous
end joining (NHEJ) pathway or (ii) the high-fidelity but less efficient homology-directed
repair (HDR) pathway (Jiang and Doudna 2017). The double-stranded DNA breaks that
are repaired by NHEJ pathway result in premature stop codon within the ORF of the
target gene by creating either deletions or insertions or frameshift mutations (Jiang and
Doudna 2017). The desired result is a loss-of-function mutation within the target gene
(Jiang and Doudna 2017). More recently, CRISPR/CAS interference method is devel-
oped, which utilizes a small guide RNA that directs the enzymatically inactive CAS
endonuclease to specific gene target resulting in interference in the transcription
(Marraffini and Sontheimer 2010). Recently, Choudhary et al. showed that by
employing an optimized CRISPR/CAS interference system, complete repression of
individual or multiple target genes in mycobacteria could be achieved, thus providing
a simple, rapid and cost-effective tool for the selective loss of gene expression in
mycobacteria (Choudhary et al. 2015). In another such study, prevention of expression
of several essential M. tuberculosis genes including pknB was reported emphasizing the
ability of the system to modulate the extent of transcription inhibition (Singh et al. 2016).
d. Immunotherapeutic Approach
With an aim to shorten the treatment duration, newer strategies are being
employed like the use of vaccines as an adjunct to standard chemotherapy.
The basic rationale behind this approach is that vaccines may have the ability to
alter the immune responses from unprotective Th2 type to protective Th1 type,
which are typically needed for M. tuberculosis control. Hence, by administering the
vaccine along with chemotherapy, it is believed that the combined effect exerted by
both together might help in faster clearance of the bacteria from the host, which can
have implications in reducing the duration of standard chemotherapy. For instance,
immunotherapy with DNA expressing α-crystallin, an antigen associated with
latency, was able to significantly reduce the chemotherapy period when compared
with the chemotherapy alone (Chauhan et al. 2013). In another study, therapeutic
vaccination of ID93/GLA-SE as an adjunct to chemotherapy decreased the bacillary
load as well as improved the survival time of mice, when compared with mice that
were given chemotherapy alone suggesting the possible benefits of adjunctive
immunotherapy in shortening the treatment time (Coler et al. 2013). Vaccination
with multivalent DNA vaccine encoding Ag85B, MPT-64 and MPT-83 in combina-
tion with isoniazid and pyrazinamide was effective in prevention of TB reactivation
(Yu et al. 2008). Silva et al. demonstrated that immunotherapy with plasmid DNA
encoding Mycobacterium leprae 65 kDa heat-shock protein (hsp65) in association
with chemotherapy shortens the duration of treatment, improves the treatment of
latent TB infection and is also effective against MDR-TB (Silva et al. 2005).
Figure 25.3 shows the problems and possible solutions involved in the TB drug
discovery program.
488 G. Khare et al.
Fig. 25.3 TB drug discovery program: problems and possible solutions
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Repurposing of Carbapenems
for the Treatment of Drug-Resistant 26
Tuberculosis
Pankaj Kumar, Urvashi B. Singh, Gyanu Lamichhane,

and Elizabeth Story-Roller
Abstract
The World Health Organization (WHO) estimated that there were 1.6 million
deaths worldwide in 2017 from tuberculosis (TB). Today, TB kills more humans
than any other infectious agent, even surpassing HIV/AIDS or malaria (WHO TB
report 2018). Prolonged treatment with poorly tolerated drugs to treat drug-
resistant tuberculosis (DR-TB) has further exacerbated this global health crisis.
Overall success rate of treating multidrug-resistant (MDR) and extensively drug-
resistant (XDR) TB is only 54%, which means for a significant number of
patients, especially those with XDR-TB, prognosis is death. Factors that make
existing treatment options of MDR/XDR-TB inadequate are (1) high incidence of
undesirable side effects from the only drugs that are available today to treat these
infections, (2) the protracted treatment duration (more than 18 months) and (3) the
high cost of the treatment. The WHO has approved several new and repurposing
of existing antibiotics for treatment of DR-TB aimed at improving outcomes of
MDR/XDR-TB treatment. These recommendations have focused on optimizing
all aspects of TB treatment including shortening treatment duration, optimizing
dosages and developing new combination regimens. Although new anti-TB
P. Kumar (*)
Department of Biochemistry, School of Chemical and Life Sciences, Jamia Hamdard, New Delhi,
Delhi, India
e-mail: pankajkumar@jamiahamdard.ac.in
U. B. Singh
Tuberculosis Section, Department of Microbiology, A.I.I.M.S., New Delhi, Delhi, India
G. Lamichhane · E. Story-Roller
Division of Infectious Diseases, School of Medicine, Johns Hopkins University, Baltimore, MD,
USA

https://doi.org/10.1007/978-981-32-9413-4_26
498 P. Kumar et al.
drugs, like bedaquiline, delamanid and pretomanid, have been successfully

developed, their slow implementation, suboptimal availability and unanticipated
cardiac toxicities mean availability of additional antibiotics would provide
choices and improve our ability to treat DR-TB. Repurposing of already available
antibiotics that are safe and readily accessible can be a practical solution for
treating DR-TB, especially in resource-poor settings where majority of TB
patients live. One class of antibiotics that matches this profile are the powerful
carbapenems of the β-lactam group. There is a long history of their use in treating
bacterial infections, they are safe and widely available, and emerging evidence
shows that they can be immediately repurposed to treat DR-TB. This book
chapter describes recent important developments in the repurposing of
carbapenems for MDR and XDR-TB treatment.
Keywords
Tuberculosis · Drug resistance · Drug repurposing · Carbapenems
Abbreviations

TB Tuberculosis
MDR Multidrug resistant
XDR Extensively drug resistant
CrfA Carbapenem-resistant factor A
PBP Penicillin-binding protein
DHP-1 Dehydropeptidase 1
26.1 Introduction
According to the most recent WHO report, global tuberculosis (TB) is one of the top
ten causes of death worldwide (Organization 2018). In 2017 alone, 10 million people
fell ill with TB and 1.6 million succumbed to death. Two-thirds of TB cases were in
ten countries alone: India (27%), China (9%), Indonesia (8%), the Philippines (6%),
Pakistan (5%), Nigeria (4%), Bangladesh (4%) and South Africa (3%). About 1.7
billion people, 23% of the world’s population, are estimated to harbour latent TB
infection and are thus at risk of developing active TB disease during their lifetime
(Organization 2018). Global prevalence of multidrug-resistant (MDR) TB and
emergence of extensively drug-resistant (XDR) TB has further elevated the severity
of this public health crisis. Three countries India (24%), China (13%) and Russian
Federation (10%) account for almost half of the world’s cases of MDR/rifampin-
resistant TB. Amongst the cases of MDR-TB in 2017, 8.5% were estimated to have
XDR-TB. The WHO has classified two additional classes of emerging drug-resistant
26 Repurposing of Carbapenems for the Treatment of Drug-Resistant Tuberculosis 499
TB, extremely drug-resistant TB (XXDR-TB) and totally drug-resistant TB

(TDR-TB) (Organization 2018). In 2016, the WHO reported that 58 countries and
territories were treating 7234 XDR-TB patients with the majority of cases notified
from four countries: India (2130), Ukraine (1206), the Russian Federation (1205)
and South Africa (719) (Organization 2016; Prasad et al. 2017). A cross-sectional
survey conducted amongst adults and children receiving antiretroviral therapy
(ART) in Mumbai, India, revealed that DR-TB was diagnosed in 68 (34%) of
202 culture-positive cases. Of these 68 cases, 38% were MDR-TB, 21% were
pre-XDR (MDR-TB plus resistance to either a fluoroquinolone or a second-line
injectable drug), 6% XDR-TB and 2% XXDR-TB (Isaakidis et al. 2014). Therefore,
there is an urgent need to develop new drugs or drug regimens to address the
challenges associated with DR-TB.
Major efforts made in the past decade to develop new therapeutics against TB
have delivered three new antibiotics, bedaquiline, delamanid and pretomanid, for TB
treatment (Zumla et al. 2015; Wong et al. 2013; Andries et al. 2005; Diacon et al.
2009; Matsumoto et al. 2006; Singh et al. 2008). However, it is still unclear if these
drugs will be safe for long-term use as is required for treating DR-TB and whether
they would be affordable to TB patients, the majority of whom are financially
destitute. An appealing solution could be repurposing of existing antibiotics that
have not been used to treat TB, but exhibit potency against it. In pursuit of this
approach, several existing drugs (sulfamethoxazole, sulfadiazine, linezolid,
minocycline, amoxicillin/clavulanate and carbapenems such as meropenem, etc.)
are currently being investigated for their efficacies to treat DR-TB (Sharma et al.
2017; Silva et al. 2018). Below we summarize recent developments in repurposing
of carbapenems to treat DR-TB.
26.2 Oldie but Goldie: ß-Lactam Antibiotic for TB Treatment
One of the antibiotic classes for which there is empirical evidence of successful use
in treating DR-TB is the β-lactams (Keener 2014). There are five major subclasses of
β-lactams, penicillins, cephalosporins, monobactams, carbapenems and penems, that
are available today for treating bacterial infections (Fig. 26.1). The order of these
β-lactam subclasses as written above also represents the chronology of their devel-
opment and introduction to the pharmaceutical market. The discovery and commer-
cial development of penicillins was a major event in the modern antibiotic era.
Beginning in the 1940s, penicillin was widely adopted to treat bacterial infections.
However, penicillins exhibited poor activity against M. tuberculosis, the bacterium
that causes TB. The existence of native β-lactamase that hydrolyses penicillins made
them ineffective against M. tuberculosis. Later when cephalosporins were devel-
oped, similar lack of activity against M. tuberculosis and their hydrolysis by
β-lactamase was documented. Based on these observations, it was considered that
antibiotics bearing the β-lactam core would be susceptible to M. tuberculosis
β-lactamase and therefore inefficacious against TB. While this concept is generally
true with penicillin and cephalosporin subclasses, emerging evidence shows that this
500 P. Kumar et al.
Fig. 26.1 Chemical structure of ß-lactam antibiotic: Penicillin, Cephalosporin, Carbapenem and
Penem
may not be applicable to newer subclasses of β-lactams, namely, carbapenems and

penems. Recent biochemical studies of M. tuberculosis and β-lactams have shown
that carbapenems and penems are not readily hydrolysed by its β-lactamase. In
addition, and perhaps more importantly, these β-lactams are uniquely effective at
inhibiting an alternate enzyme class with which M. tuberculosis generates its cell
wall peptidoglycan (Kumar et al. 2017b; Correale et al. 2013), a structural molecule
that is required for survival, growth and cell division.
β-Lactams exert their activity by inhibiting synthesis of peptidoglycan. The final
step of peptidoglycan synthesis involves polymerization of stem peptides to generate
a single three-dimensional molecule that becomes the peptidoglycan. According to
the historical model, only one enzyme class, namely, D,D-transpeptidases (also
known as penicillin-binding proteins), catalyses this reaction (Walsh 2003)). This
model of peptidoglycan was developed using rapidly growing model bacteria such
as B. subtilis and E. coli. While this model is representative of many bacteria, recent
discoveries demonstrate that peptidoglycan of M. tuberculosis is unique, and the
final step of peptidoglycan synthesis is undertaken by two distinct enzyme classes:
the D,D-transpeptidases and the newly identified L,D-transpeptidases (Erdemli et al.
2012; Gupta et al. 2010; Brammer Basta et al. 2015; Schoonmaker et al. 2014; Walsh
2003). In M. tuberculosis, the L,D-transpeptidases catalyse higher proportions of the
reaction and therefore are potentially likely to be at least as important as the D,
D-transpeptidases (Gupta et al. 2010). Recent studies have shown that, unlike
penicillins and cephalosporins, carbapenems and penems are effective at inhibiting
L,D-transpeptidases of M. tuberculosis (Kumar et al. 2017b; Brammer Basta et al.
2015; Erdemli et al. 2012; Gupta et al. 2010; Moraes et al. 2015; Correale et al. 2013;
Kim et al. 2013). These studies suggest that higher potencies of carbapenems and
penems against M. tuberculosis are likely based not only on their ability to inhibit D,
D-transpeptidases and resist hydrolysis by β-lactamase (Hugonnet et al. 2009) but
more critically because of their unique ability to inhibit the L,D-transpeptidases
(Fig. 26.2).
Assessment of minimum inhibitory concentrations confirmed that carbapenems
and penems are more potent than penicillins and cephalosporins against
Fig. 26.2 Carbapenem inhibits multiple targets in Mycobacterium tuberculosis. BlaC ß-lactamase,
PBP Penicillin Binding Protein, CrfA Carbapenem Resistance Factor A
502 P. Kumar et al.
M. tuberculosis (Hugonnet et al. 2009; Kaushik et al. 2015; Solapure et al. 2013;
Horita et al. 2014). Carbapenems also inhibit a new class of ß-lactam hydrolysing
enzymes in M. tuberculosis, carbapenem-resistant factor A (CrfA), which can confer
M. tuberculosis resistance to carbapenems (Kumar et al. 2017b).
To date, meropenem, imipenem and ertapenem have been tested for synergy with
clavulanate in XDR-TB patients through intravenous route of administration.
Faropenem, a penem, has been clinically tested in a limited number of patients in
combination with other TB drugs (Silva et al. 2018; Arbex et al. 2016; Tiberi et al.
2016a, b, c; van Rijn et al. 2016; Zuur et al. 2018; Payen et al. 2018; Diacon et al.
2016; Deshpande et al. 2016). There is no published data on the clinical use of
doripenem, panipenem or biapenem (Zhang et al. 2016), but their efficacies have
been studied in vitro or in vivo in preclinical models of TB (Kaushik et al. 2015;
Kumar et al. 2017a, b; Kaushik et al. 2017a, b; Story-Roller and Lamichhane 2018;
Walsh 2003).
26.3 Empirical Evidence of Efficacy of Carbapenems Against TB
Clinicians have evaluated the potential utility of ß-lactams for treating MDR and
XDR-TB. Anecdotally, in select MDR-TB patients, regimens containing amoxicil-
lin/clavulanate and other second-line agents have been effective against MDR-TB,
though this approach has met considerable scepticism (Yew and Chau 1996).
Thereafter, different subclasses of ß-lactams, including various penicillins,
cephalosporins and carbapenems, have been anecdotally tried for the treatment of
MDR and XDR-TB (Ramon-Garcia et al. 2016; Jaganath et al. 2016; Chambers et al.
2005). Amongst the different subclasses of ß-lactam drugs, only carbapenems have
emerged as potent inhibitors of MDR-TB and XDR-TB. In 2005, clinical efficacy of
imipenem was tested in ten patients in conjunction with an aminoglycoside or
capreomycin, which indicated that imipenem was therapeutically useful for TB
treatment (Chambers et al. 2005). Thereafter, several carbapenems have been tested
for their clinical efficacy in MDR-TB and XDR-TB settings (Sotgiu et al. 2015a, b,
Sotgiu et al. 2016; Jaganath et al. 2016) and are currently in various stages of clinical
trials in combination with other anti-TB drugs as seen in Table 26.1.
26.4 Repurposing of Meropenem
After Hugonnet et al. (Hugonnet et al. 2009) demonstrated in a laboratory setting that
the carbapenem drug meropenem, combined with clavulanate, is consistently effec-
tive against XDR-TB, there has been tremendous progress in the repurposing of
carbapenems for MDR/XDR-TB treatment. Soon after this seminal publication, an
in vivo study evaluated the efficacy of meropenem/clavulanate in a chronically
infected mouse model of TB (England et al. 2012). In this study, one group of
mice was treated with meropenem alone at 300 mg/kg body weight by subcutaneous
injection twice daily, and a second group was given the same dosage of meropenem
Table 26.1 Different clinical trials of carbapenems for treatment of drug-resistant TB

Clinical trial Sponsor Clinical study
Drug identifier institute phase status Title of study
Rifampin + NCT03174184 Johns 2 (recruiting) A Phase 2a study of
Meropenem + Hopkins the early
Amoxicillin/ University bactericidal activity
Clavulanate of Rifampin (RIF)
in combination with
Meropenem Plus
Amoxicillin/
Clavulanate among
adults with
Rifampin-resistant
or Rifampin-
susceptible
pulmonary
Tuberculosis
Meropenem + NCT02349841 Task 2 (completed) A Phase 2 trial to
Amoxiycillin/ Foundation evaluate the early
clavulanic NPC bactericidal activity,
Faropenem, safety and
Amoxiycillin/ tolerability of
clavulanic acid Meropenem,
administered
intravenously, Plus
Amoxiycillin/CA
and Faropenem,
administered orally,
Plus Amoxiycillin/
CA in adult patients
with newly
diagnosed, smear-
positive pulmonary
Tuberculosis.
Rifampicin, NCT03237182 Centre for 4 (recruiting) The individualized
rifabutin, isoniazid, the AIDS M(X) drug-resistant
high dose isoniazid, Programme TB treatment
pyrazinamide, of Research strategy study A
ethambutol, in strategy to improve
levofloxacin, South Africa treatment outcomes
moxifloxacin, in patients with
ofloxacin, drug-resistant TB
gatifloxacin,
amikacin,
capreomycin,
kanamycin,
streptomycin,
ethionamide,
prothionamide,
cycloserine,
terizidone,
pretomanid,
(continued)
504 P. Kumar et al.

linezolid, sutezolid,
clofazimine,
bedaquiline,
delamainid, para-
aminosalicylic acid,
imipenem/cilastatin,
Meropenem,
amoxicillin/
clavulanate,
clarithromycin,
azithromycin and
thioacetazone
Isoniazid, NCT03625739 Beijing (recruiting) Population
Rifampicin, Children's Pharmacokinetics
Pyrazinamide Hospital of antituberculosis
Ethambutol, drugs in children
Levofloxacin, with Tuberculosis
Moxifloxacin,
Gatifloxacin,
Amikacin,
Capreomycin,
Kanamycin
(Streptomycin),
Ethionamide,
Cycloserine,
terizidone,
Clofazimine,
Bedaquiline,
Delamanid,
p-aminosalicylic
acid, Imipenem-
cilastatin,
Amoxicillin-
clavulanat e,
Thioacetazone
Ertapenem NCT01730664 University 2 (completed) Pharmacokinetics
Medical and
Center Pharmacodynamics
Groningen of Ertapenem in
patients with
Tuberculosis
Faropenem, NCT02393586 National 1 (completed) Evaluating
Amoxicillin/ University pharmacokinetics
clavulanic acid Hospital, and whole blood
500 mg/125 mg Singapore bactericidal activity
Rifampicin 10 mg/ against
kg Mycobacterium
Tuberculosis of
single doses of
(continued)

Faropenem Plus
Amoxicillin/
Clavulanic acid in
healthy volunteers
Faropenem, NCT02381470 National 2 (not yet Trial of Faropenem
Amoxicillin/ University recruiting) and Cefadroxil
clavulanic acid Hospital, (in combination
500 mg/125 mg Singapore with Amoxicillin/
cCefadroxil 1 g Clavulanic Acid
Rifampicin 10 mg/ and standard TB
kg drugs) in patients
with pulmonary
Tuberculosis :
measurement of
early bactericidal
activity and effects
on novel
biomarkers
in combination with 50 mg/kg of clavulanate given orally. Both meropenem alone

and the combination of meropenem with clavulanate significantly reduced the
bacterial burden in both the spleen and lungs. These drugs were also tested in
New Zealand white rabbits, which are capable of developing hypoxic pulmonary
lesions. To prevent hydrolysis of meropenem by renal dihydropeptidase-1 (DHP-1),
which is much more active in rabbits compared to humans, the DHP-1 inhibitor
cilastatin was co-administered with meropenem. Cilastatin increased drug exposure
by 4-fold, but resulted in significant adverse effects leading to early cessation of the
experiment (England et al. 2012). In summary, meropenem in synergy with
clavulanate exhibits bactericidal activity in macrophage and murine models of TB,
but assessment in higher animals such as rabbits is complicated by the increased
activity of renal DHP-1 that hydrolyses carbapenems. Since the human isoform of
DHP-1 is less active than in mice or rabbits, prospective clinical evaluation of
meropenem/clavulanate in humans with chronic tuberculosis was absolutely
warranted.
Following the discovery of potency of meropenem/clavulanate against TB in
in vitro and in vivo models, a clinical trial (n ¼ 96) evaluating the effectiveness and
safety of meropenem-/clavulanate-containing regimens against MDR-TB and
XDR-TB was performed (Tiberi et al. 2016b). In this study, similar sputum culture
conversion rates were observed between meropenem-/clavulanate-containing and
meropenem-/clavulanate-sparing regimens against XDR-TB; however, treatment
success in the XDR-TB cohort with meropenem/clavulanate was higher than previ-
ously achieved in the largest observational cohort study of MDR/XDR-TB treatment
to date (46.8% vs. 40%) (Falzon et al. 2013). In another study that evaluated early
bactericidal activity of meropenem in combination with amoxicillin and clavulanate
506 P. Kumar et al.
in TB patients, this combination exhibited desirable bactericidal activity

(NCCT02349841) (Diacon et al. 2016).
Another phase 2 clinical trial is underway for evaluating the early bactericidal
efficacy of rifampin in combination with meropenem plus amoxicillin/clavulanate
amongst adults with rifampin-resistant TB (NCT03174184). A phase 4 clinical trial,
sponsored by the Centre for the AIDS Programme of Research in South Africa
(NCT03237182), is currently in progress to develop an individualized treatment
strategy for drug-resistant tuberculosis. This trial aims to use M. tuberculosis whole
genome sequencing prior to selection of a drug-resistant tuberculosis treatment
regimen that may include various drugs, including meropenem. Results from all
three clinical trial studies on meropenem are currently pending (Table 26.1).
Recently, a retrospective observational case series study was published assessing
long-term follow-up of XDR-TB patients treated with meropenem/clavulanate
(Payen et al. 2018). A total of 18 patients were followed for a median of four
years after TB treatment. TB isolates from these patients were resistant to a median
of 10 anti-tubercular drugs; 15 isolates were XDR-TB and three were pre-XDR. Out
of 18, 15 patients achieved cure, and no relapses occurred in these patients on a
median follow-up of 4 years (Payen et al. 2018). No adverse events were observed
with meropenem/clavulanate, but the use of implantable intravenous device leads to
catheter infection resulting in septicaemia in six patients.
26.5 Repurposing of Imipenem
Imipenem and meropenem were initially assessed for their in vitro antibacterial
activity against M. tuberculosis by Watt et al. in 1992 (Watt et al. 1992). The first
clinical evaluation of imipenem efficacy against TB included ten patients with TB
isolates resistant to both isoniazid and rifampin (Chambers et al. 2005). In this study,
patients were treated with imipenem in addition to standard first- or second-line anti-
tubercular drugs. Eight of 10 patients achieved sputum culture conversion and seven
patients remained culture negative off treatment, suggesting imipenem as a potential
adjunctive treatment option for MDR-TB.
One clinical trial sponsored by Beijing Children’s Hospital (NCT03625739,
Table 26.1) is currently recruiting patients for the study of pharmacokinetics of
anti-TB drugs, including imipenem, in children with TB.
A comparative study of imipenem/clavulanate and meropenem/clavulanate has
also been performed to compare their relative efficacies against TB (Tiberi et al.
2016c). Eighty-four patients with MDR/XDR-TB were treated with imipenem/
clavulanate, and 96 patients were treated with meropenem/clavulanate in conjunc-
tion with other TB drugs. The cohort of patients treated with a meropenem-/
clavulanate-containing TB regimen achieved better results compared to imipenem/
clavulanate. Specifically, culture conversion rates and treatment success rates were
significantly higher with meropenem/clavulanate (Tiberi et al. 2016b). Both
meropenem/clavulanate and imipenem/clavulanate were well tolerated with minimal
adverse effects. Imipenem, however, is cheaper than meropenem and is more
economically viable in developing countries. Imipenem/cilastatin is now categorized

by WHO as a group 5 medication for the treatment of MDR/XDR-TB.
26.6 Repurposing of Ertapenem
The first clinical research of ertapenem for TB treatment was published in 2016
(Tiberi et al. 2016a). In vitro and pharmacological data suggested that ertapenem has
a longer half-life than meropenem, allowing a more simplified single parenteral
administration of ertapenem per day compared to thrice daily administration of
meropenem or imipenem (Tremblay et al. 2010; Tiberi et al. 2016a). In this small
retrospective cohort study, five patients with pulmonary TB (two patients with XDR
and three with pre-XDR) had been treated with regimens containing ertapenem. Of
these, four patients achieved cure and ertapenem was generally well tolerated. A
phase 2 clinical trial studying pharmacokinetics and pharmacodynamics of
ertapenem in patients with TB has recently been completed by the University
Medical Center Groningen (NCT01730664, Table 26.1).
26.7 Repurposing of Faropenem
Faropenem is an orally bioavailable penem that has shown efficacy against TB both
in vitro and in vivo, with superior bactericidal activity compared to meropenem
(Dhar et al. 2015). Additional in vitro efficacy studies of several carbapenems, in
addition to faropenem, against TB showed that faropenem was the most potent
single agent after tebipenem against M. tuberculosis H37Rv strain (Kumar et al.
2017b). Faropenem was also noted to exhibit significant synergy when combined
with rifampin against H37Rv as well as several clinical TB isolates (Kaushik et al.
2015). Of note, this synergistic effect was also observed to a lesser extent between
rifampin and carbapenems doripenem, biapenem and meropenem.
Two clinical trials studying faropenem have been completed: a phase 1 trial by
the National University Hospital, Singapore (NCT02393586), and a phase 2 trial by
TASK Foundation NPC (NCT02349841). Both studies evaluated the anti-tubercular
activity and pharmacokinetics of faropenem in synergy with amoxicillin/clavulanate.
Additionally, the National University Hospital, Singapore, is conducting a phase
2 trial of faropenem/cefadroxil in combination with amoxicillin/clavulanate
(NCT02381470). Results of the first trial have been published by Gurumurthy
et al., in which blood samples from healthy volunteers were inoculated with Mtb
H37Rv shortly after treatment with either rifampin alone, faropenem plus amoxicil-
lin/clavulanate or a combination of all three drugs. They found that while faropenem
plus amoxicillin/clavulanate exhibited no bactericidal activity, the three-drug com-
bination was effective (Gurumurthy et al. 2017). Of note, this is in contrast to the
previously cited study by Dhar et al. that showed efficacy of faropenem alone against
an intracellular macrophage model of TB (Dhar et al. 2015).
508 P. Kumar et al.
Most recently, in 2018, 20 clinical isolates, including MDR and XDR, were tested
for susceptibility against ertapenem and faropenem. Eighteen out of twenty isolates
were resistant to ertapenem (in combination with amoxicillin/clavulanate). 50% of
the isolates showed some level of susceptibility against faropenem that was further
augmented in combination with amoxicillin/clavulanate (Giovanni Satta 2018).
26.8 Repurposing of Biapenem
Biapenem has been shown to exhibit bactericidal activity against M. tuberculosis

in vitro and in vivo (Kaushik et al. 2017a, b, 2015; Kumar et al. 2017a, b; Zhang
et al. 2016), and an in vivo study in mice directly comparing biapenem to faropenem
against TB showed both agents to reduce bacterial loads in the lungs compared to
untreated controls, with biapenem exhibiting superior killing. In this study, biapenem
was found to be effective against both H37Rv and rifampin-resistant strains. The
addition of rifampin resulted in further synergistic activity against H37Rv, although
this was not observed amongst resistant strains (Kumar et al. 2017b).
To date, there is not a single clinical study on biapenem for the treatment of TB,
but preclinical studies suggest this drug warrants further investigation. This is
especially true in light of the fact that biapenem is much more resistant to hydrolysis
by renal dehydropeptidase 1 (DHP-1) than meropenem, which may make it a more
viable agent in vivo (Hikida et al. 1992).
26.9 Repurposing of Tebipenem
Although few studies evaluating tebipenem against TB have been performed, it

does appear to exhibit anti-tubercular activity in vitro, with increased efficacy noted
with the addition of the β-lactamase inhibitor, clavulanate (Horita et al. 2014;
Kaushik et al. 2015).
26.10 Development of Evolved Carbapenems Against

Tuberculosis
The ß-lactam antibiotics, especially carbapenems, are being repurposed for the
treatment of drug-resistant tuberculosis. However, these antibiotics were developed
to treat other non-tubercular infections. There is a need to understand the molecular
mechanism of inhibition of M. tuberculosis by ß-lactams and develop new ß-lactams
that exhibit improved efficacy against M. tuberculosis. An initiative of such kind was
taken where new evolved carbapenems were developed based on structure-based
drug designing by targeting a newly discovered class of L,D-transpeptidase enzymes
that are involved in synthesis of 3 !3 trans-peptide linkages in the peptidoglycan of
M. tuberculosis (Kumar et al. 2017b).
Fig. 26.3 Crystal structure of L,D-transpeptidase 2 (LdtMt2) in covalent complex with newly
evolved anti-TB carbapenem drugs. (a) Crystal structure with T206; (b) with T208 and (c) with
T210. (Kumar et al. 2017a, b)
Based on structure-activity relationship data (SAR), 12 evolved carbapenems

were reported to show in vitro antibacterial activity with minimum inhibitory
concentration (MIC90) lower than that of meropenem, doripenem, biapenem,
faropenem and tebipenem against M. tuberculosis. Four of synthesized evolved
carbapenems, T205, T206, T208 and T210, were further evaluated for molecular
interaction studies with L,D-transpeptidases LdtMt1 and LdtMt2. These compounds
exhibited irreversible inhibition of the L,D-transpeptidases. Crystal structures of
T206, T208 and T210 were solved in complex with L,D-transpeptidase LdtMt2
(Fig. 26.3) (Kumar et al. 2017b). Crystal structures of evolved carbapenems with
L,D-transpeptidase will be valuable for further development of more therapeutically
potent carbapenems that are also specific against M. tuberculosis.
26.11 Future Prospects in the Use of Carbapenems for TB

Treatment
ß-Lactam antibiotics are the most commonly used antibiotics worldwide, yet only
recently have we developed an appreciation for their anti-tubercular activity (espe-
cially carbapenems) and the potentiating effects of ß-lactamase inhibitors like
510 P. Kumar et al.
clavulanate. This class of antibiotics has been re-evaluated in recent years due to the
shortage of new drugs for the treatment of DR-TB. There is limited preclinical and
clinical data on the use of carbapenems in combination with conventional anti-TB
drugs. More rigorous in vivo evaluations should be undertaken to identify optimized
dosing strategies to treat DR-TB. Most of the existing carbapenems are administered
intravenously, which is impractical in the clinical setting, especially in resource-
limited areas. Particular emphasis should be placed on development of oral
carbapenems, as both of the currently available oral carbapenem and penem
formulations, tebipenem and faropenem, respectively, have shown potency against
M. tuberculosis.
A coordinated effort is necessary in Asian, Southeast Asian and African countries
that harbour the highest number of TB cases worldwide. These countries are in
desperate need of novel drugs and combinations capable of effectively treating drug-
resistant TB if we hope to eradicate this pathogen. Clinical trials assessing
combinations of carbapenems with conventional anti-TB drugs for treatment of
MDR-TB and XDR-TB have been undertaken in the USA and Europe, where
carbapenem cost is high and TB prevalence is low. Countries with high TB burdens
such as India and China have the potential to develop carbapenems at low cost and
can easily enrol patients in clinical trials and consequently accelerate research and
development of potent carbapenem containing regimens to treat DR-TB.
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Mycobacterium Tuberculosis: Molecular: Infection Biology, Pathogenesis, Diagnostics and New Interventions

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ISBN 978-981-32-9412-7 ISBN 978-981-32-9413-4 (eBook)

# Springer Nature Singapore Pte Ltd. 2019

Pathogenesis and Epidemiology of Tuberculosis

Molecular and Infection Biology of M.tb

Vaccines, Diagnostics, and New Interventions

New Delhi, Delhi, India Seyed Ehtesham Hasnain

Part I Pathogenesis and Epidemiology of Tuberculosis

Part II Molecular and Infection Biology of M. tuberculosis

10 Comparative In Silico Analyses Reveal Crucial Factors

Part III Vaccines, Diagnostics and New Interventions

21 Tuberculosis Vaccine: Past Experiences and Future Prospects . . . . 375

Prof. Seyed Ehtesham Hasnain has contributed signiﬁcantly to tuberculo-

Dr. Sonam Grover is a UGC Assistant Professor at JH Institute of Molecular

Fahad Abdullah Department of Respiratory Medicine, Deccan College of Medical

Rakesh Bhatnagar Molecular Biology and Genetic Engineering Laboratory,

Seyed Ehtesham Hasnain JH Institute of Molecular Medicine, Jamia Hamdard,

Prachi Nangpal Department of Biochemistry, University of Delhi South Campus,

Jyoti Sharma Department of Biotechnology and Bioinformatics, School of Life

Anil K. Tyagi Department of Biochemistry, University of Delhi South Campus,

That which is not expressed will be forgotten.

BCG Bacillus Calmette-Guerin

# Springer Nature Singapore Pte Ltd. 2019 3

MDR-TB Multidrug-resistant tuberculosis

Slightly differential versions of the following story exist: but essentially, it

In 1908 Charles Mantoux’s method of injecting tuberculin directly into the

In 1943, the tide turned. Twenty-three-year-old Albert Schatz joined Waksman’s

Two new anti-TB drugs—bedaquiline and delamanid—have ﬁnally broken

# Springer Nature Singapore Pte Ltd. 2019 17

AIDS acquired immunodeﬁciency syndrome

Tuberculosis (TB) is a major cause of infectious disease mortality worldwide, killing

Compression of the bronchi by enlarged

Tuberculosis Intrinsic obstructive and stenotic lesions

1.67 million deaths in 2016 est. COPD

Incidence rate of 140 / 100,000 est. Pulmonary TB can lead to a remodelling

This results in a deterioration in lung

2.2 TB and Bronchiectasis

bronchiectasis in adult TB results from intrinsic obstructive and stenotic lesions of

2.3 TB and COPD

Chronic obstructive pulmonary disease (COPD) refers to chronic airﬂow obstruction

cavitation, traction bronchiectasis, bronchostenosis, or parenchymal lung destruction

2.4 Conclusions and Future Perspectives

Epidemiological associations are emerging between communicable and

molecular levels. An improved level of understanding of the interactions between

Funding Not applicable.

Ethical Approval Not required.

Geldmacher C, Ngwenyama N, Schuetz A, Petrovas C, Reither K, Heeregrave EJ, Casazza JP,

Nag V, Nandan D, Singh S, Arora S, Tsanglao W, Arya N (2015) Pulmonary tuberculosis: a

AIDS Acquired immunodeﬁciency syndrome

# Springer Nature Singapore Pte Ltd. 2019 27

HIV Human immunodeﬁciency virus

3.2 Gender Differences

In general, most studies have reported a signiﬁcantly higher occurrence of TB in men

3.2.1 Socioeconomic Factors

availability of healthcare services, poor living conditions such as ventilation of

3.3 Smoking and Alcohol

A meta-analysis of 24 studies by Bates et al. showed that smoking is a risk factor

3.4 Diabetes Mellitus

3.6 Immunocompromised Patients

3.7 Immune Therapeutics and Susceptibility to TB

mycobacteria which in turn can lead to reactivation of TB (Cantini et al. 2018).

# Springer Nature Singapore Pte Ltd. 2019 37

successful treatment outcome, active TB Drug-Safety Monitoring and Manage-